Antisense modulation of LAR expression

ABSTRACT

Antisense compounds, compositions and methods are provided for modulating the expression of LAR. The compositions comprise antisense compounds, particularly antisense oligonucleotides, targeted to nucleic acids encoding LAR. Methods of using these compounds for modulation of LAR expression and for treatment of diseases associated with expression of LAR are provided.

FIELD OF THE INVENTION

[0001] The present invention provides compositions and methods formodulating the expression of LAR. In particular, this invention relatesto compounds, particularly oligonucleotides, specifically hybridizablewith nucleic acids encoding LAR. Such compounds have been shown tomodulate the expression of LAR.

BACKGROUND OF THE INVENTION

[0002] The process of phosphorylation, defined as the attachment of aphosphate moiety to a biological molecule through the action of enzymescalled kinases, represents one course by which intracellular signals arepropagated resulting finally in a cellular response. Within the cell,proteins can be phosphorylated on serine, threonine or tyrosine residuesand the extent of phosphorylation is regulated by the opposing action ofphosphatases, which remove the phosphate moieties. While the majority ofprotein phosphorylation within the cell is on serine and threonineresidues, tyrosine phosphorylation is modulated to the greatest extentduring oncogenic transformation and growth factor stimulation (Zhang,Critical Review in Biochemistry and Molecular Biology, 1998, 33, 1-52).

[0003] Because phosphorylation is such a ubiquitous process within cellsand because cellular phenotypes are largely influenced by the activityof these pathways, it is currently believed that a number of diseasestates and/or disorders are a result of either aberrant activation of,or functional mutations in, kinases and phosphatases. Consequently,considerable attention has been devoted recently to the characterizationof tyrosine kinases and tyrosine phosphatases.

[0004] Leukocyte antigen-related phosphatase (LAR, also known as proteintyrosine phosphatase, receptor type F; PTPRF and LCA-homolog) is aprototype for a family of transmembrane phosphatases whose extracellularregions are composed of a combination of immunoglobulin-like domains andfibronectin type III (Fn-III) domains (Streuli et al., Embo J., 1992,11, 897-907.; Streuli et al., J. Exp. Med., 1988, 168, 1523-1530). LARwas first cloned in 1988 and mapped to chromosome 1p32-33 in 1992(Streuli et al., Embo J., 1992, 11, 897-907.; Streuli et al., J. Exp.Med., 1988, 168, 1523-1530). It is expressed in cells of many differentlineages including epithelial cells, smooth muscle cells and cardiacmyocytes (Streuli et al., Embo J., 1992, 11, 897-907.). It issynthesized as a precursor with a molecular weight above 200 kDa whichis cleaved by an endogenous protease into two subunits (150 and 85 kDa)that remain non-covalently attached (Streuli et al., Embo J., 1992, 11,897-907.).

[0005] Alternative splicing of LAR has been observed in sections of mRNAencoding the FN-III domains 4, 5, 6 and 7 in various combinations(O'Grady et al., J. Biol. Chem., 1994, 269, 25193-25199). Analternatively-spliced 11 amino acid proximal membrane segment of LAR(LAR alternatively spliced element-a; LASE-a) has been shown tocontribute to regulation of LAR expression and function during neuritedevelopment (Honkaniemi et al., Brain Res. Mol. Brain Res., 1998, 60,1-12). Increased levels of LAR expression and differential patterns ofextracellular alternative splicing were found in breast cancer celllines and pheochromocytoma tumor tissue (Yang et al., Carcinogenesis,2000, 21, 125-131; Yang et al., Mol. Carcinog., 1999, 25, 139-149).

[0006] LAR has emerged as an important candidate enzyme for theregulation of the insulin receptor because it is widely expressed ininsulin-sensitive tissues and its cytoplasmic domain has a catalyticpreference for the regulatory phosphotyrosines of the insulin receptorkinase domain in vitro (Goldstein et al., Mol. Cell. Biochem., 1998,182, 91-99).

[0007] Overexpression of LAR in a variety of mammalian cells inducescell death without affecting cell adhesion. This suggests that LAR mayactivate the caspase pathway and induce cell death directly (Weng etal., Curr. Biol., 1998, 8, 247-256).

[0008] Enhanced expression of LAR has been observed in corneal cellswith keratoconus, a corneal thinning disorder that leads to irregularastigmatism and corneal distortion (Chiplunkar et al., Exp. Eye Res.,1999, 68, 283-293).

[0009] The involvement of LAR in cell signaling events make it apotentially useful therapeutic target for intervention inhyperproliferative disorders, metabolic disorders and disorders arisingfrom aberrant apoptosis.

[0010] Small molecule inhibitors of tyrosine phosphatases exist in theart. For example, disclosed and claimed in U.S. Pat. No. 6,169,087 aresmall molecule inhibitors of protein tyrosine phosphatases for thetreatment of type I diabetes, type II diabetes, impaired glucosetolerance, insulin resistance, obesity, and a number of other diseases(Andersen et al., 2001).

[0011] Disclosed and claimed in PCT publication WO 00/61180 is a methodfor identifying indolinones, quinazolines, quinoxalines and tyrphostinsthat modulate LAR activity (Ullrich and Muller, 2000).

[0012] O'Grady et al. have shown that anti-LAR antibodies inhibit theinteraction of LAR with the laminin-nidogen complex and have determinedthat binding of LAR to laminin-nidogen may play a role in regulatingcell signaling because inhibition of this interaction causes cellmorphological changes (O'Grady et al., J. Cell Biol., 1998, 141,1675-1684).

[0013] LAR antisense vectors have been used to inhibit LAR ininvestigations of the effects of LAR expression on insulin receptorsignaling and apolipoprotein B metabolism in rat hepatoma cells (Kulaset al., J. Biol. Chem., 1995, 270, 2435-2438.; Mooney et al., Biochem.Biophys. Res. Commun., 1997, 235, 709-712; Phung et al., Biochem.Biophys. Res. Commun., 1997, 237, 367-371) and apoptosis in rat PC12cells (Tisi et al., J. Neurobiol., 2000, 42, 477-486).

[0014] To date, investigative strategies aimed at modulating LARfunction have involved the use of small molecule inhibitors, antibodiesand antisense LAR vectors. However, these strategies have yet to betested as therapeutic protocols. Consequently, there remains a long feltneed for agents capable of effectively inhibiting LAR function.

[0015] Antisense technology is emerging as an effective means forreducing the expression of specific gene products and may thereforeprove to be uniquely useful in a number of therapeutic, diagnostic, andresearch applications for the modulation of LAR expression.

[0016] The present invention provides compositions and methods formodulating LAR expression.

SUMMARY OF THE INVENTION

[0017] The present invention is directed to compounds, particularlyantisense oligonucleotides, which are targeted to a nucleic acidencoding LAR, and which modulate the expression of LAR. Pharmaceuticaland other compositions comprising the compounds of the invention arealso provided. Further provided are methods of modulating the expressionof LAR in cells or tissues comprising contacting said cells or tissueswith one or more of the antisense compounds or compositions of theinvention. Further provided are methods of treating an animal,particularly a human, suspected of having or being prone to a disease orcondition associated with expression of LAR by administering atherapeutically or prophylactically effective amount of one or more ofthe antisense compounds or compositions of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0018] The present invention employs oligomeric compounds, particularlyantisense oligonucleotides, for use in modulating the function ofnucleic acid molecules encoding LAR, ultimately modulating the amount ofLAR produced. This is accomplished by providing antisense compoundswhich specifically hybridize with one or more nucleic acids encodingLAR. As used herein, the terms “target nucleic acid” and “nucleic acidencoding LAR” encompass DNA encoding LAR, RNA (including pre-mRNA andmRNA) transcribed from such DNA, and also cDNA derived from such RNA.The specific hybridization of an oligomeric compound with its targetnucleic acid interferes with the normal function of the nucleic acid.This modulation of function of a target nucleic acid by compounds whichspecifically hybridize to it is generally referred to as “antisense”.The functions of DNA to be interfered with include replication andtranscription. The functions of RNA to be interfered with include allvital functions such as, for example, translocation of the RNA to thesite of protein translation, translocation of the RNA to sites withinthe cell which are distant from the site of RNA synthesis, translationof protein from the RNA, splicing of the RNA to yield one or more mRNAspecies, and catalytic activity which may be engaged in or facilitatedby the RNA. The overall effect of such interference with target nucleicacid function is modulation of the expression of LAR. In the context ofthe present invention, “modulation” means either an increase(stimulation) or a decrease (inhibition) in the expression of a gene. Inthe context of the present invention, inhibition is the preferred formof modulation of gene expression and mRNA is a preferred target.

[0019] It is preferred to target specific nucleic acids for antisense.“Targeting” an antisense compound to a particular nucleic acid, in thecontext of this invention, is a multistep process. The process usuallybegins with the identification of a nucleic acid sequence whose functionis to be modulated. This may be, for example, a cellular gene (or mRNAtranscribed from the gene) whose expression is associated with aparticular disorder or disease state, or a nucleic acid molecule from aninfectious agent. In the present invention, the target is a nucleic acidmolecule encoding LAR. The targeting process also includes determinationof a site or sites within this gene for the antisense interaction tooccur such that the desired effect, e.g., detection or modulation ofexpression of the protein, will result. Within the context of thepresent invention, a preferred intragenic site is the regionencompassing the translation initiation or termination codon of the openreading frame (ORF) of the gene. Since, as is known in the art, thetranslation initiation codon is typically 5′-AUG (in transcribed mRNAmolecules; 5′-ATG in the corresponding DNA molecule), the translationinitiation codon is also referred to as the “AUG codon,” the “startcodon” or the “AUG start codon”. A minority of genes have a translationinitiation codon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Thus, theterms “translation initiation codon” and “start codon” can encompassmany codon sequences, even though the initiator amino acid in eachinstance is typically methionine (in eukaryotes) or formylmethionine (inprokaryotes). It is also known in the art that eukaryotic andprokaryotic genes may have two or more alternative start codons, any oneof which may be preferentially utilized for translation initiation in aparticular cell type or tissue, or under a particular set of conditions.In the context of the invention, “start codon” and “translationinitiation codon” refer to the codon or codons that are used in vivo toinitiate translation of an mRNA molecule transcribed from a geneencoding LAR, regardless of the sequence(s) of such codons.

[0020] It is also known in the art that a translation termination codon(or “stop codon”) of a gene may have one of three sequences, i.e.,5′-UAA, 5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA,5′-TAG and 5′-TGA, respectively). The terms “start codon region” and“translation initiation codon region” refer to a portion of such an mRNAor gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationinitiation codon. Similarly, the terms “stop codon region” and“translation termination codon region” refer to a portion of such anmRNA or gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationtermination codon.

[0021] The open reading frame (ORF) or “coding region,” which is knownin the art to refer to the region between the translation initiationcodon and the translation termination codon, is also a region which maybe targeted effectively. Other target regions include the 5′untranslated region (5′UTR), known in the art to refer to the portion ofan mRNA in the 5′ direction from the translation initiation codon, andthus including nucleotides between the 5′ cap site and the translationinitiation codon of an mRNA or corresponding nucleotides on the gene,and the 3′ untranslated region (3′UTR), known in the art to refer to theportion of an mRNA in the 3′ direction from the translation terminationcodon, and thus including nucleotides between the translationtermination codon and 3′ end of an mRNA or corresponding nucleotides onthe gene. The 5′ cap of an mRNA comprises an N7-methylated guanosineresidue joined to the 5′-most residue of the mRNA via a 5′-5′triphosphate linkage. The 5′ cap region of an mRNA is considered toinclude the 5′ cap structure itself as well as the first 50 nucleotidesadjacent to the cap. The 5′ cap region may also be a preferred targetregion.

[0022] Although some eukaryotic mRNA transcripts are directlytranslated, many contain one or more regions, known as “introns,” whichare excised from a transcript before it is translated. The remaining(and therefore translated) regions are known as “exons” and are splicedtogether to form a continuous mRNA sequence. mRNA splice sites, i.e.,intron-exon junctions, may also be preferred target regions, and areparticularly useful in situations where aberrant splicing is implicatedin disease, or where an overproduction of a particular mRNA spliceproduct is implicated in disease. Aberrant fusion junctions due torearrangements or deletions are also preferred targets. mRNA transcriptsproduced via the process of splicing of two (or more) mRNAs fromdifferent gene sources are known as “fusion transcripts”. It has alsobeen found that introns can be effective, and therefore preferred,target regions for antisense compounds targeted, for example, to DNA orpre-mRNA.

[0023] It is also known in the art that alternative RNA transcripts canbe produced from the same genomic region of DNA. These alternativetranscripts are generally known as “variants”. More specifically,“pre-mRNA variants” are transcripts produced from the same genomic DNAthat differ from other transcripts produced from the same genomic DNA ineither their start or stop position and contain both intronic andextronic regions.

[0024] Upon excision of one or more exon or intron regions or portionsthereof during splicing, pre-mRNA variants produce smaller “mRNAvariants”. Consequently, mRNA variants are processed pre-mRNA variantsand each unique pre-mRNA variant must always produce a unique mRNAvariant as a result of splicing. These mRNA variants are also known as“alternative splice variants”. If no splicing of the pre-mRNA variantoccurs then the pre-mRNA variant is identical to the mRNA variant.

[0025] It is also known in the art that variants can be produced throughthe use of alternative signals to start or stop transcription and thatpre-mRNAs and mRNAs can possess more that one start codon or stop codon.Variants that originate from a pre-mRNA or mRNA that use alternativestart codons are known as “alternative start variants” of that pre-mRNAor mRNA. Those transcripts that use an alternative stop codon are knownas “alternative stop variants” of that pre-mRNA or mRNA. One specifictype of alternative stop variant is the “polyA variant” in which themultiple transcripts produced result from the alternative selection ofone of the “polyA stop signals” by the transcription machinery, therebyproducing transcripts that terminate at unique polyA sites.

[0026] Once one or more target sites have been identified;oligonucleotides are chosen which are sufficiently complementary to thetarget, i.e., hybridize sufficiently well and with sufficientspecificity, to give the desired effect.

[0027] In the context of this invention, “hybridization” means hydrogenbonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary nucleoside or nucleotide bases.For example, adenine and thymine are complementary nucleobases whichpair through the formation of hydrogen bonds. “Complementary,” as usedherein, refers to the capacity for precise pairing between twonucleotides. For example, if a nucleotide at a certain position of anoligonucleotide is capable of hydrogen bonding with a nucleotide at thesame position of a DNA or RNA molecule, then the oligonucleotide and theDNA or RNA are considered to be complementary to each other at thatposition. The oligonucleotide and the DNA or RNA are complementary toeach other when a sufficient number of corresponding positions in eachmolecule are occupied by nucleotides which can hydrogen bond with eachother. Thus, “specifically hybridizable” and “complementary” are termswhich are used to indicate a sufficient degree of complementarity orprecise pairing such that stable and specific binding occurs between theoligonucleotide and the DNA or RNA target. It is understood in the artthat the sequence of an antisense compound need not be 100%complementary to that of its target nucleic acid to be specificallyhybridizable.

[0028] An antisense compound is specifically hybridizable when bindingof the compound to the target DNA or RNA molecule interferes with thenormal function of the target DNA or RNA to cause a loss of activity,and there is a sufficient degree of complementarity to avoidnon-specific binding of the antisense compound to non-target sequencesunder conditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, and in the case of in vitro assays, under conditions in whichthe assays are performed. It is preferred that the antisense compoundsof the present invention comprise at least 80% sequence complementarityto a target region within the target nucleic acid, moreover that theycomprise 90% sequence complementarity and even more comprise 95%sequence complementarity to the target region within the target nucleicacid sequence to which they are targeted. For example, an antisensecompound in which 18 of 20 nucleobases of the antisense compound arecomplementary, and would therefore specifically hybridize, to a targetregion would represent 90 percent complementarity. Percentcomplementarity of an antisense compound with a region of a targetnucleic acid can be determined routinely using basic local alignmentsearch tools (BLAST programs) (Altschul et al., J. Mol. Biol., 1990,215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).

[0029] Antisense and other compounds of the invention, which hybridizeto the target and inhibit expression of the target, are identifiedthrough experimentation, and representative sequences of these compoundsare hereinbelow identified as preferred embodiments of the invention.The sites to which these preferred antisense compounds are specificallyhybridizable are hereinbelow referred to as “preferred target regions”and are therefore preferred sites for targeting. As used herein the term“preferred target region” is defined as at least an 8-nucleobase portionof a target region to which an active antisense compound is targeted.While not wishing to be bound by theory, it is presently believed thatthese target regions represent regions of the target nucleic acid whichare accessible for hybridization.

[0030] While the specific sequences of particular preferred targetregions are set forth below, one of skill in the art will recognize thatthese serve to illustrate and describe particular embodiments within thescope of the present invention. Additional preferred target regions maybe identified by one having ordinary skill.

[0031] Target regions 8-80 nucleobases in length comprising a stretch ofat least eight (8) consecutive nucleobases selected from within theillustrative preferred target regions are considered to be suitablepreferred target regions as well.

[0032] Exemplary good preferred target regions include DNA or RNAsequences that comprise at least the 8 consecutive nucleobases from the5′-terminus of one of the illustrative preferred target regions (theremaining nucleobases being a consecutive stretch of the same DNA or RNAbeginning immediately upstream of the 5′-terminus of the target regionand continuing until the DNA or RNA contains about 8 to about 80nucleobases). Similarly good preferred target regions are represented byDNA or RNA sequences that comprise at least the 8 consecutivenucleobases from the 3′-terminus of one of the illustrative preferredtarget regions (the remaining nucleobases being a consecutive stretch ofthe same DNA or RNA beginning immediately downstream of the 3′-terminusof the target region and continuing until the DNA or RNA contains about8 to about 80 nucleobases). One having skill in the art, once armed withthe empirically-derived preferred target regions illustrated herein willbe able, without undue experimentation, to identify further preferredtarget regions. In addition, one having ordinary skill in the art willalso be able to identify additional compounds, including oligonucleotideprobes and primers, that specifically hybridize to these preferredtarget regions using techniques available to the ordinary practitionerin the art.

[0033] Antisense compounds are commonly used as research reagents anddiagnostics. For example, antisense oligonucleotides, which are able toinhibit gene expression with exquisite specificity, are often used bythose of ordinary skill to elucidate the function of particular genes.Antisense compounds are also used, for example, to distinguish betweenfunctions of various members of a biological pathway. Antisensemodulation has, therefore, been harnessed for research use.

[0034] For use in kits and diagnostics, the antisense compounds of thepresent invention, either alone or in combination with other antisensecompounds or therapeutics, can be used as tools in differential and/orcombinatorial analyses to elucidate expression patterns of a portion orthe entire complement of genes expressed within cells and tissues.

[0035] Expression patterns within cells or tissues treated with one ormore antisense compounds are compared to control cells or tissues nottreated with antisense compounds and the patterns produced are analyzedfor differential levels of gene expression as they pertain, for example,to disease association, signaling pathway, cellular localization,expression level, size, structure or function of the genes examined.These analyses can be performed on stimulated or unstimulated cells andin the presence or absence of other compounds which affect expressionpatterns.

[0036] Examples of methods of gene expression analysis known in the artinclude DNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000,480, 17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serialanalysis of gene expression) (Madden, et al., Drug Discov. Today, 2000,5, 415-425), READS (restriction enzyme amplification of digested cDNAs)(Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (totalgene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci. U.S. A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, et al.,FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis, 1999,20, 2100-10), expressed sequence tag (EST) sequencing (Celis, et al.,FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000, 80,143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal.Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41,203-208), subtractive cloning, differential display (DD) (Jurecic andBelmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomichybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31,286-96), FISH (fluorescent in situ hybridization) techniques (Going andGusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometrymethods (reviewed in To, Comb. Chem. High Throughput Screen, 2000, 3,235-41).

[0037] The specificity and sensitivity of antisense is also harnessed bythose of skill in the art for therapeutic uses. Antisenseoligonucleotides have been employed as therapeutic moieties in thetreatment of disease states in animals and man. Antisenseoligonucleotide drugs, including ribozymes, have been safely andeffectively administered to humans and numerous clinical trials arepresently underway. It is thus established that oligonucleotides can beuseful therapeutic modalities that can be configured to be useful intreatment regimes for treatment of cells, tissues and animals,especially humans.

[0038] In the context of this invention, the term “oligonucleotide”refers to an oligomer or polymer of ribonucleic acid (RNA) ordeoxyribonucleic acid (DNA) or mimetics thereof. This term includesoligonucleotides composed of naturally-occurring nucleobases, sugars andcovalent internucleoside (backbone) linkages as well as oligonucleotideshaving non-naturally-occurring portions which function similarly. Suchmodified or substituted oligonucleotides are often preferred over nativeforms because of desirable properties such as, for example, enhancedcellular uptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases.

[0039] While antisense oligonucleotides are a preferred form ofantisense compound, the present invention comprehends other oligomericantisense compounds, including but not limited to oligonucleotidemimetics such as are described below. The antisense compounds inaccordance with this invention preferably comprise from about 8 to about80 nucleobases (i.e. from about 8 to about 80 linked nucleosides).Particularly preferred antisense compounds are antisenseoligonucleotides from about 8 to about 50 nucleobases, even morepreferably those comprising from about 12 to about 30 nucleobases.Antisense compounds include ribozymes, external guide sequence (EGS)oligonucleotides (oligozymes), and other short catalytic RNAs orcatalytic oligonucleotides which hybridize to the target nucleic acidand modulate its expression.

[0040] Antisense compounds 8-80 nucleobases in length comprising astretch of at least eight (8) consecutive nucleobases selected fromwithin the illustrative antisense compounds are considered to besuitable antisense compounds as well.

[0041] Exemplary preferred antisense compounds include DNA or RNAsequences that comprise at least the 8 consecutive nucleobases from the5′-terminus of one of the illustrative preferred antisense compounds(the remaining nucleobases being a consecutive stretch of the same DNAor RNA beginning immediately upstream of the 5′-terminus of theantisense compound which is specifically hybridizable to the targetnucleic acid and continuing until the DNA or RNA contains about 8 toabout 80 nucleobases). Similarly preferred antisense compounds arerepresented by DNA or RNA sequences that comprise at least the 8consecutive nucleobases from the 3′-terminus of one of the illustrativepreferred antisense compounds (the remaining nucleobases being aconsecutive stretch of the same DNA or RNA beginning immediatelydownstream of the 3′-terminus of the antisense compound which isspecifically hybridizable to the target nucleic acid and continuinguntil the DNA or RNA contains about 8 to about 80 nucleobases). Onehaving skill in the art, once armed with the empirically-derivedpreferred antisense compounds illustrated herein will be able, withoutundue experimentation, to identify further preferred antisensecompounds.

[0042] Antisense and other compounds of the invention, which hybridizeto the target and inhibit expression of the target, are identifiedthrough experimentation, and representative sequences of these compoundsare herein identified as preferred embodiments of the invention. Whilespecific sequences of the antisense compounds are set forth herein, oneof skill in the art will recognize that these serve to illustrate anddescribe particular embodiments within the scope of the presentinvention. Additional preferred antisense compounds may be identified byone having ordinary skill.

[0043] As is known in the art, a nucleoside is a base-sugar combination.The base portion of the nucleoside is normally a heterocyclic base. Thetwo most common classes of such heterocyclic bases are the purines andthe pyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. In turn, the respective ends of this linearpolymeric structure can be further joined to form a circular structure,however, open linear structures are generally preferred. In addition,linear structures may also have internal nucleobase complementarity andmay therefore fold in a manner as to produce a double strandedstructure. Within the oligonucleotide structure, the phosphate groupsare commonly referred to as forming the internucleoside backbone of theoligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′to 5′ phosphodiester linkage.

[0044] Specific examples of preferred antisense compounds useful in thisinvention include oligonucleotides containing modified backbones ornon-natural internucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

[0045] Preferred modified oligonucleotide backbones include, forexample, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotri-esters,methyl and other alkyl phosphonates including 3′-alkylene phosphonates,5′-alkylene phosphonates and chiral phosphonates, phosphinates,phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphatesand borano-phosphates having normal 3′-5′ linkages, 2′-5′ linked analogsof these, and those having inverted polarity wherein one or moreinternucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.Preferred oligonucleotides having inverted polarity comprise a single 3′to 3′ linkage at the 3′-most internucleotide linkage i.e. a singleinverted nucleoside residue which may be abasic (the nucleobase ismissing or has a hydroxyl group in place thereof). Various salts, mixedsalts and free acid forms are also included.

[0046] Representative United States patents that teach the preparationof the above phosphorus-containing linkages include, but are not limitedto, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243;5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717;5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677;5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253;5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218;5,672,697 and 5,625,050, certain of which are commonly owned with thisapplication, and each of which is herein incorporated by reference.

[0047] Preferred modified oligonucleotide backbones that do not includea phosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts.

[0048] Representative United States patents that teach the preparationof the above oligonucleosides include, but are not limited to, U.S. Pat.Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain ofwhich are commonly owned with this application, and each of which isherein incorporated by reference.

[0049] In other preferred oligonucleotide mimetics, both the sugar andthe internucleoside linkage, i.e., the backbone, of the nucleotide unitsare replaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative U.S. patents that teach thepreparation of PNA compounds include, but are not limited to, U.S. Pat.Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 1991, 254, 1497-1500.

[0050] Most preferred embodiments of the invention are oligonucleotideswith phosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [knownas a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereferenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

[0051] Modified oligonucleotides may also contain one or moresubstituted sugar moieties. Preferred oligonucleotides comprise one ofthe following at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, orN-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred areO[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃,O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃]₂, where n and m are from1 to about 10. Other preferred oligonucleotides comprise one of thefollowing at the 2′ position: C₁ to C₁₀ lower alkyl, substituted loweralkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH,SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of anoligonucleotide, or a group for improving the pharmacodynamic propertiesof an oligonucleotide, and other substituents having similar properties.A preferred modification includes 2′-methoxyethoxy (2′—O—CH₂CH₂OCH₃,also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv.Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A furtherpreferred modification includes 2′-dimethylaminooxyethoxy, i.e., aO(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in exampleshereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e.,2′—O—CH₂—O—CH₂—N(CH₃)₂, also described in examples hereinbelow.

[0052] Other preferred modifications include 2′-methoxy (2′—O—CH₃),2′-aminopropoxy (2′—OCH₂CH₂CH₂NH₂), 2′-allyl (2′—CH₂—CH═CH₂), 2′-O-allyl(2′—O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modification may be inthe arabino (up) position or ribo (down) position. A preferred2′-arabino modification is 2′-F. Similar modifications may also be madeat other positions on the oligonucleotide, particularly the 3′ positionof the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide.Oligonucleotides may also have sugar mimetics such as cyclobutylmoieties in place of the pentofuranosyl sugar. Representative UnitedStates patents that teach the preparation of such modified sugarstructures include, but are not limited to, U.S. Pat. Nos. 4,981,957;5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786;5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909;5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633;5,792,747; and 5,700,920, certain of which are commonly owned with theinstant application, and each of which is herein incorporated byreference in its entirety.

[0053] A further preferred modification includes Locked Nucleic Acids(LNAs) in which the 2′-hydroxyl group is linked to the 3′ or 4′ carbonatom of the sugar ring thereby forming a bicyclic sugar moiety. Thelinkage is preferably a methelyne (—CH₂—)_(n) group bridging the 2′oxygen atom and the 4′ carbon atom wherein n is 1 or 2. LNAs andpreparation thereof are described in WO 98/39352 and WO 99/14226.

[0054] Oligonucleotides may also include nucleobase (often referred toin the art simply as “base”) modifications or substitutions. As usedherein, “unmodified” or “natural” nucleobases include the purine basesadenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C) and uracil (U). Modified nucleobases include othersynthetic and natural nucleobases such as 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl(—C≡C—CH₃) uracil and cytosine and other alkynyl derivatives ofpyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modifiednucleobases include tricyclic pyrimidines such as phenoxazinecytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobasesmay also include those in which the purine or pyrimidine base isreplaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, pages858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosedby Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, pages 289-302, Crooke, S. T. and Lebleu, B.ed., CRC Press, 1993. Certain of these nucleobases are particularlyuseful for increasing the binding affinity of the oligomeric compoundsof the invention. These include 5-substituted pyrimidines,6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. andLebleu, B., eds., Antisense Research and Applications, CRC Press, BocaRaton, 1993, pp. 276-278) and are presently preferred basesubstitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

[0055] Representative United States patents that teach the preparationof certain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and5,681,941, certain of which are commonly owned with the instantapplication, and each of which is herein incorporated by reference, andU.S. Pat. No. 5,750,692, which is commonly owned with the instantapplication and also herein incorporated by reference.

[0056] Another modification of the oligonucleotides of the inventioninvolves chemically linking to the oligonucleotide one or more moietiesor conjugates which enhance the activity, cellular distribution orcellular uptake of the oligonucleotide. The compounds of the inventioncan include conjugate groups covalently bound to functional groups suchas primary or secondary hydroxyl groups. Conjugate groups of theinvention include intercalators, reporter molecules, polyamines,polyamides, polyethylene glycols, polyethers, groups that enhance thepharmacodynamic properties of oligomers, and groups that enhance thepharmacokinetic properties of oligomers. Typical conjugate groupsinclude cholesterols, lipids, phospholipids, biotin, phenazine, folate,phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines,coumarins, and dyes. Groups that enhance the pharmacodynamic properties,in the context of this invention, include groups that improve oligomeruptake, enhance oligomer resistance to degradation, and/or strengthensequence-specific hybridization with RNA. Groups that enhance thepharmacokinetic properties, in the context of this invention, includegroups that improve oligomer uptake, distribution, metabolism orexcretion. Representative conjugate groups are disclosed inInternational Patent Application PCT/US92/09196, filed Oct. 23, 1992 theentire disclosure of which is incorporated herein by reference.Conjugate moieties include but are not limited to lipid moieties such asa cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA,1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem.Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharanet al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphaticchain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al.,EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259,327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid,e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937). Oligonucleotides of the invention mayalso be conjugated to active drug substances, for example, aspirin,warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen,(S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoicacid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide,a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug,an antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drugconjugates and their preparation are described in U.S. patentapplication Ser. No. 09/334,130 (filed Jun. 15, 1999) which isincorporated herein by reference in its entirety.

[0057] Representative United States patents that teach the preparationof such oligonucleotide conjugates include, but are not limited to, U.S.Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313;5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584;5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439;5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779;4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013;5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136;5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873;5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475;5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481;5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941,certain of which are commonly owned with the instant application, andeach of which is herein incorporated by reference.

[0058] It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide. The present invention alsoincludes antisense compounds which are chimeric compounds. “Chimeric”antisense compounds or “chimeras,” in the context of this invention, areantisense compounds, particularly oligonucleotides, which contain two ormore chemically distinct regions, each made up of at least one monomerunit, i.e., a nucleotide in the case of an oligonucleotide compound.These oligonucleotides typically contain at least one region wherein theoligonucleotide is modified so as to confer upon the oligonucleotideincreased resistance to nuclease degradation, increased cellular uptake,increased stability and/or increased binding affinity for the targetnucleic acid. An additional region of the oligonucleotide may serve as asubstrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. Byway of example, RNAse H is a cellular endonuclease which cleaves the RNAstrand of an RNA:DNA duplex. Activation of RNase H, therefore, resultsin cleavage of the RNA target, thereby greatly enhancing the efficiencyof oligonucleotide inhibition of gene expression. The cleavage ofRNA:RNA hybrids can, in like fashion, be accomplished through theactions of endoribonucleases, such as interferon-induced RNAseL whichcleaves both cellular and viral RNA. Consequently, comparable resultscan often be obtained with shorter oligonucleotides when chimericoligonucleotides are used, compared to phosphorothioatedeoxyoligonucleotides hybridizing to the same target region. Cleavage ofthe RNA target can be routinely detected by gel electrophoresis and, ifnecessary, associated nucleic acid hybridization techniques known in theart.

[0059] Chimeric antisense compounds of the invention may be formed ascomposite structures of two or more oligonucleotides, modifiedoligonucleotides, oligonucleosides and/or oligonucleotide mimetics asdescribed above. Such compounds have also been referred to in the art ashybrids or gapmers. Representative United States patents that teach thepreparation of such hybrid structures include, but are not limited to,U.S. Pat. Nos.: 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878;5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and5,700,922, certain of which are commonly owned with the instantapplication, and each of which is herein incorporated by reference inits entirety.

[0060] The antisense compounds used in accordance with this inventionmay be conveniently and routinely made through the well-known techniqueof solid phase synthesis. Equipment for such synthesis is sold byseveral vendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

[0061] The compounds of the invention may also be admixed, encapsulated,conjugated or otherwise associated with other molecules, moleculestructures or mixtures of compounds, as for example, liposomes,receptor-targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.Representative United States patents that teach the preparation of suchuptake, distribution and/or absorption-assisting formulations include,but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

[0062] The antisense compounds of the invention encompass anypharmaceutically acceptable salts, esters, or salts of such esters, orany other compound which, upon administration to an animal, including ahuman, is capable of providing (directly or indirectly) the biologicallyactive metabolite or residue thereof. Accordingly, for example, thedisclosure is also drawn to prodrugs and pharmaceutically acceptablesalts of the compounds of the invention, pharmaceutically acceptablesalts of such prodrugs, and other bioequivalents.

[0063] The term “prodrug” indicates a therapeutic agent that is preparedin an inactive form that is converted to an active form (i.e., drug)within the body or cells thereof by the action of endogenous enzymes orother chemicals and/or conditions. In particular, prodrug versions ofthe oligonucleotides of the invention are prepared as SATE[(S-acetyl-2-thioethyl) phosphate] derivatives according to the methodsdisclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 orin WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al.

[0064] The term “pharmaceutically acceptable salts” refers tophysiologically and pharmaceutically acceptable salts of the compoundsof the invention: i.e., salts that retain the desired biologicalactivity of the parent compound and do not impart undesiredtoxicological effects thereto.

[0065] Pharmaceutically acceptable base addition salts are formed withmetals or amines, such as alkali and alkaline earth metals or organicamines. Examples of metals used as cations are sodium, potassium,magnesium, calcium, and the like. Examples of suitable amines areN,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine(see, for example, Berge et al., “Pharmaceutical Salts,” J. of PharmaSci., 1977, 66, 1-19). The base addition salts of said acidic compoundsare prepared by contacting the free acid form with a sufficient amountof the desired base to produce the salt in the conventional manner. Thefree acid form may be regenerated by contacting the salt form with anacid and isolating the free acid in the conventional manner. The freeacid forms differ from their respective salt forms somewhat in certainphysical properties such as solubility in polar solvents, but otherwisethe salts are equivalent to their respective free acid for purposes ofthe present invention. As used herein, a “pharmaceutical addition salt”includes a pharmaceutically acceptable salt of an acid form of one ofthe components of the compositions of the invention. These includeorganic or inorganic acid salts of the amines. Preferred acid salts arethe hydrochlorides, acetates, salicylates, nitrates and phosphates.Other suitable pharmaceutically acceptable salts are well known to thoseskilled in the art and include basic salts of a variety of inorganic andorganic acids, such as, for example, with inorganic acids, such as forexample hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoricacid; with organic carboxylic, sulfonic, sulfo or phospho acids orN-substituted sulfamic acids, for example acetic acid, propionic acid,glycolic acid, succinic acid, maleic acid, hydroxymaleic acid,methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid,oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, salicylic acid,4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid,embonic acid, nicotinic acid or isonicotinic acid; and with amino acids,such as the 20 alpha-amino acids involved in the synthesis of proteinsin nature, for example glutamic acid or aspartic acid, and also withphenylacetic acid, methanesulfonic acid, ethanesulfonic acid,2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,benzenesulfonic acid, 4-methylbenzenesulfonic acid,naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (withthe formation of cyclamates), or with other acid organic compounds, suchas ascorbic acid. Pharmaceutically acceptable salts of compounds mayalso be prepared with a pharmaceutically acceptable cation. Suitablepharmaceutically acceptable cations are well known to those skilled inthe art and include alkaline, alkaline earth, ammonium and quaternaryammonium cations. Carbonates or hydrogen carbonates are also possible.

[0066] For oligonucleotides, preferred examples of pharmaceuticallyacceptable salts include but are not limited to (a) salts formed withcations such as sodium, potassium, ammonium, magnesium, calcium,polyamines such as spermine and spermidine, etc.; (b) acid additionsalts formed with inorganic acids, for example hydrochloric acid,hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and thelike; (c) salts formed with organic acids such as, for example, aceticacid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaricacid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoicacid, tannic acid, palmitic acid, alginic acid, polyglutamic acid,naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d)salts formed from elemental anions such as chlorine, bromine, andiodine.

[0067] The antisense compounds of the present invention can be utilizedfor diagnostics, therapeutics, prophylaxis and as research reagents andkits. For therapeutics, an animal, preferably a human, suspected ofhaving a disease or disorder which can be treated by modulating theexpression of LAR is treated by administering antisense compounds inaccordance with this invention. The compounds of the invention can beutilized in pharmaceutical compositions by adding an effective amount ofan antisense compound to a suitable pharmaceutically acceptable diluentor carrier. Use of the antisense compounds and methods of the inventionmay also be useful prophylactically, e.g., to prevent or delayinfection, inflammation or tumor formation, for example.

[0068] The antisense compounds of the invention are useful for researchand diagnostics, because these compounds hybridize to nucleic acidsencoding LAR, enabling sandwich and other assays to easily beconstructed to exploit this fact. Hybridization of the antisenseoligonucleotides of the invention with a nucleic acid encoding LAR canbe detected by means known in the art. Such means may includeconjugation of an enzyme to the oligonucleotide, radiolabelling of theoligonucleotide or any other suitable detection means. Kits using suchdetection means for detecting the level of LAR in a sample may also beprepared.

[0069] The present invention also includes pharmaceutical compositionsand formulations which include the antisense compounds of the invention.The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic and to mucousmembranes including vaginal and rectal delivery), pulmonary, e.g., byinhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal), oralor parenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial, e.g., intrathecal or intraventricular,administration. Oligonucleotides with at least one 2′-O-methoxyethylmodification are believed to be particularly useful for oraladministration.

[0070] Pharmaceutical compositions and formulations for topicaladministration may include transdermal patches, ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable. Coated condoms,gloves and the like may also be useful. Preferred topical formulationsinclude those in which the oligonucleotides of the invention are inadmixture with a topical delivery agent such as lipids, liposomes, fattyacids, fatty acid esters, steroids, chelating agents and surfactants.Preferred lipids and liposomes include neutral (e.g.dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl cholineDMPC, distearolyphosphatidyl choline) negative (e.g.dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). Oligonucleotides of the invention may beencapsulated within liposomes or may form complexes thereto, inparticular to cationic liposomes. Alternatively, oligonucleotides may becomplexed to lipids, in particular to cationic lipids. Preferred fattyacids and esters include but are not limited arachidonic acid, oleicacid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristicacid, palmitic acid, stearic acid, linoleic acid, linolenic acid,dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or aC₁₋₁₀ alkyl ester (e.g. isopropylmyristate IPM), monoglyceride,diglyceride or pharmaceutically acceptable salt thereof. Topicalformulations are described in detail in U.S. patent application Ser. No.09/315,298 filed on May 20, 1999 which is incorporated herein byreference in its entirety.

[0071] Compositions and formulations for oral administration includepowders or granules, microparticulates, nanoparticulates, suspensions orsolutions in water or non-aqueous media, capsules, gel capsules,sachets, tablets or minitablets. Thickeners, flavoring agents, diluents,emulsifiers, dispersing aids or binders may be desirable. Preferred oralformulations are those in which oligonucleotides of the invention areadministered in conjunction with one or more penetration enhancerssurfactants and chelators. Preferred surfactants include fatty acidsand/or esters or salts thereof, bile acids and/or salts thereof.Preferred bile acids/salts include chenodeoxycholic acid (CDCA) andursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid,deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid,taurocholic acid, taurodeoxycholic acid, sodiumtauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Preferredfatty acids include arachidonic acid, undecanoic acid, oleic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or amonoglyceride, a diglyceride or a pharmaceutically acceptable saltthereof (e.g. sodium). Also preferred are combinations of penetrationenhancers, for example, fatty acids/salts in combination with bileacids/salts. A particularly preferred combination is the sodium salt oflauric acid, capric acid and UDCA. Further penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether.Oligonucleotides of the invention may be delivered orally, in granularform including sprayed dried particles, or complexed to form micro ornanoparticles. Oligonucleotide complexing agents include poly-aminoacids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes,polyalkylcyanoacrylates; cationized gelatins, albumins, starches,acrylates, polyethyleneglycols (PEG) and starches;polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans,celluloses and starches. Particularly preferred complexing agentsinclude chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine,polyornithine, polyspermines, protamine, polyvinylpyridine,polythiodiethylamino-methylethylene P(TDAE), polyaminostyrene (e.g.p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulationsfor oligonucleotides and their preparation are described in detail inU.S. application Ser. Nos. 08/886,829 (filed Jul. 1, 1997), 09/108,673(filed Jul. 1, 1998), 09/256,515 (filed Feb. 23, 1999), 09/082,624(filed May 21, 1998) and 09/315,298 (filed May 20, 1999), each of whichis incorporated herein by reference in their entirety.

[0072] Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

[0073] Pharmaceutical compositions of the present invention include, butare not limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

[0074] The pharmaceutical formulations of the present invention, whichmay conveniently be presented in unit dosage form, may be preparedaccording to conventional techniques well known in the pharmaceuticalindustry. Such techniques include the step of bringing into associationthe active ingredients with the pharmaceutical carrier(s) orexcipient(s). In general, the formulations are prepared by uniformly andintimately bringing into association the active ingredients with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product.

[0075] The compositions of the present invention may be formulated intoany of many possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

[0076] In one embodiment of the present invention the pharmaceuticalcompositions may be formulated and used as foams. Pharmaceutical foamsinclude formulations such as, but not limited to, emulsions,microemulsions, creams, jellies and liposomes. While basically similarin nature these formulations vary in the components and the consistencyof the final product. The preparation of such compositions andformulations is generally known to those skilled in the pharmaceuticaland formulation arts and may be applied to the formulation of thecompositions of the present invention.

[0077] Emulsions

[0078] The compositions of the present invention may be prepared andformulated as emulsions. Emulsions are typically heterogenous systems ofone liquid dispersed in another in the form of droplets usuallyexceeding 0.1 μm in diameter (Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 2, p. 335; Higuchi et al., in Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p.301). Emulsions are often biphasic systems comprising two immiscibleliquid phases intimately mixed and dispersed with each other. Ingeneral, emulsions may be of either the water-in-oil (w/o) or theoil-in-water (o/w) variety. When an aqueous phase is finely divided intoand dispersed as minute droplets into a bulk oily phase, the resultingcomposition is called a water-in-oil (w/o) emulsion. Alternatively, whenan oily phase is finely divided into and dispersed as minute dropletsinto a bulk aqueous phase, the resulting composition is called anoil-in-water (o/w) emulsion. Emulsions may contain additional componentsin addition to the dispersed phases, and the active drug which may bepresent as a solution in either the aqueous phase, oily phase or itselfas a separate phase. Pharmaceutical excipients such as emulsifiers,stabilizers, dyes, and anti-oxidants may also be present in emulsions asneeded. Pharmaceutical emulsions may also be multiple emulsions that arecomprised of more than two phases such as, for example, in the case ofoil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.Such complex formulations often provide certain advantages that simplebinary emulsions do not. Multiple emulsions in which individual oildroplets of an o/w emulsion enclose small water droplets constitute aw/o/w emulsion. Likewise a system of oil droplets enclosed in globulesof water stabilized in an oily continuous phase provides an o/w/oemulsion.

[0079] Emulsions are characterized by little or no thermodynamicstability. Often, the dispersed or discontinuous phase of the emulsionis well dispersed into the external or continuous phase and maintainedin this form through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion may be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatmay be incorporated into either phase of the emulsion. Emulsifiers maybroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199).

[0080] Synthetic surfactants, also known as surface active agents, havefound wide applicability in the formulation of emulsions and have beenreviewed in the literature (Rieger, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York,N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic andcomprise a hydrophilic and a hydrophobic portion. The ratio of thehydrophilic to the hydrophobic nature of the surfactant has been termedthe hydrophile/lipophile balance (HLB) and is a valuable tool incategorizing and selecting surfactants in the preparation offormulations. Surfactants may be classified into different classes basedon the nature of the hydrophilic group: nonionic, anionic, cationic andamphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Riegerand Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,p. 285).

[0081] Naturally occurring emulsifiers used in emulsion formulationsinclude lanolin, beeswax, phosphatides, lecithin and acacia. Absorptionbases possess hydrophilic properties such that they can soak up water toform w/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

[0082] A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

[0083] Hydrophilic colloids or hydrocolloids include naturally occurringgums and synthetic polymers such as polysaccharides (for example,acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, andtragacanth), cellulose derivatives (for example, carboxymethylcelluloseand carboxypropylcellulose), and synthetic polymers (for example,carbomers, cellulose ethers, and carboxyvinyl polymers). These disperseor swell in water to form colloidal solutions that stabilize emulsionsby forming strong interfacial films around the dispersed-phase dropletsand by increasing the viscosity of the external phase.

[0084] Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that may readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used may be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

[0085] The application of emulsion formulations via dermatological, oraland parenteral routes and methods for their manufacture have beenreviewed in the literature (Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 199). Emulsion formulations for oral deliveryhave been very widely used because of ease of formulation, as well asefficacy from an absorption and bioavailability standpoint (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil baselaxatives, oil-soluble vitamins and high fat nutritive preparations areamong the materials that have commonly been administered orally as o/wemulsions.

[0086] In one embodiment of the present invention, the compositions ofoligonucleotides and nucleic acids are formulated as microemulsions. Amicroemulsion may be defined as a system of water, oil and amphiphilewhich is a single optically isotropic and thermodynamically stableliquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 245). Typically microemulsions are systems that areprepared by first dispersing an oil in an aqueous surfactant solutionand then adding a sufficient amount of a fourth component, generally anintermediate chain-length alcohol to form a transparent system.Therefore, microemulsions have also been described as thermodynamicallystable, isotropically clear dispersions of two immiscible liquids thatare stabilized by interfacial films of surface-active molecules (Leungand Shah, in: Controlled Release of Drugs: Polymers and AggregateSystems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages185-215). Microemulsions commonly are prepared via a combination ofthree to five components that include oil, water, surfactant,cosurfactant and electrolyte. Whether the microemulsion is of thewater-in-oil (w/o) or an oil-in-water (o/w) type is dependent on theproperties of the oil and surfactant used and on the structure andgeometric packing of the polar heads and hydrocarbon tails of thesurfactant molecules (Schott, in Remington's Pharmaceutical Sciences,Mack Publishing Co., Easton, Pa., 1985, p. 271).

[0087] The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared toconventional emulsions, microemulsions offer the advantage ofsolubilizing water-insoluble drugs in a formulation of thermodynamicallystable droplets that are formed spontaneously.

[0088] Surfactants used in the preparation of microemulsions include,but are not limited to, ionic surfactants, non-ionic surfactants, Brij96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions may, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase may typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase may include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

[0089] Microemulsions are particularly of interest from the standpointof drug solubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (Constantinideset al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm.Sci., 1996, 85, 138-143). Often microemulsions may form spontaneouslywhen their components are brought together at ambient temperature. Thismay be particularly advantageous when formulating thermolabile drugs,peptides or oligonucleotides. Microemulsions have also been effective inthe transdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of oligonucleotides and nucleic acidsfrom the gastrointestinal tract, as well as improve the local cellularuptake of oligonucleotides and nucleic acids within the gastrointestinaltract, vagina, buccal cavity and other areas of administration.

[0090] Microemulsions of the present invention may also containadditional components and additives such as sorbitan monostearate (Grill3), Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the oligonucleotides andnucleic acids of the present invention. Penetration enhancers used inthe microemulsions of the present invention may be classified asbelonging to one of five broad categories—surfactants, fatty acids, bilesalts, chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

[0091] Liposomes

[0092] There are many organized surfactant structures besidesmicroemulsions that have been studied and used for the formulation ofdrugs. These include monolayers, micelles, bilayers and vesicles.Vesicles, such as liposomes, have attracted great interest because oftheir specificity and the duration of action they offer from thestandpoint of drug delivery. As used in the present invention, the term“liposome” means a vesicle composed of amphiphilic lipids arranged in aspherical bilayer or bilayers.

[0093] Liposomes are unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior. Theaqueous portion contains the composition to be delivered. Cationicliposomes possess the advantage of being able to fuse to the cell wall.Non-cationic liposomes, although not able to fuse as efficiently withthe cell wall, are taken up by macrophages in vivo.

[0094] In order to cross intact mammalian skin, lipid vesicles must passthrough a series of fine pores, each with a diameter less than 50 nm,under the influence of a suitable transdermal gradient. Therefore, it isdesirable to use a liposome which is highly deformable and able to passthrough such fine pores.

[0095] Further advantages of liposomes include; liposomes obtained fromnatural phospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated drugs in their internal compartments frommetabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245). Important considerations in thepreparation of liposome formulations are the lipid surface charge,vesicle size and the aqueous volume of the liposomes.

[0096] Liposomes are useful for the transfer and delivery of activeingredients to the site of action. Because the liposomal membrane isstructurally similar to biological membranes, when liposomes are appliedto a tissue, the liposomes start to merge with the cellular membranesand as the merging of the liposome and cell progresses, the liposomalcontents are emptied into the cell where the active agent may act.

[0097] Liposomal formulations have been the focus of extensiveinvestigation as the mode of delivery for many drugs. There is growingevidence that for topical administration, liposomes present severaladvantages over other formulations. Such advantages include reducedside-effects related to high systemic absorption of the administereddrug, increased accumulation of the administered drug at the desiredtarget, and the ability to administer a wide variety of drugs, bothhydrophilic and hydrophobic, into the skin.

[0098] Several reports have detailed the ability of liposomes to deliveragents including high-molecular weight DNA into the skin. Compoundsincluding analgesics, antibodies, hormones and high-molecular weightDNAs have been administered to the skin. The majority of applicationsresulted in the targeting of the upper epidermis.

[0099] Liposomes fall into two broad classes. Cationic liposomes arepositively charged liposomes which interact with the negatively chargedDNA molecules to form a stable complex. The positively chargedDNA/liposome complex binds to the negatively charged cell surface and isinternalized in an endosome. Due to the acidic pH within the endosome,the liposomes are ruptured, releasing their contents into the cellcytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147,980-985).

[0100] Liposomes which are pH-sensitive or negatively-charged, entrapDNA rather than complex with it. Since both the DNA and the lipid aresimilarly charged, repulsion rather than complex formation occurs.Nevertheless, some DNA is entrapped within the aqueous interior of theseliposomes. pH-sensitive liposomes have been used to deliver DNA encodingthe thymidine kinase gene to cell monolayers in culture. Expression ofthe exogenous gene was detected in the target cells (Zhou et al.,Journal of Controlled Release, 1992, 19, 269-274).

[0101] One major type of liposomal composition includes phospholipidsother than naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

[0102] Several studies have assessed the topical delivery of liposomaldrug formulations to the skin. Application of liposomes containinginterferon to guinea pig skin resulted in a reduction of skin herpessores while delivery of interferon via other means (e.g. as a solutionor as an emulsion) were ineffective (Weiner et al., Journal of DrugTargeting, 1992, 2, 405-410). Further, an additional study tested theefficacy of interferon administered as part of a liposomal formulationto the administration of interferon using an aqueous system, andconcluded that the liposomal formulation was superior to aqueousadministration (du Plessis et al., Antiviral Research, 1992, 18,259-265).

[0103] Non-ionic liposomal systems have also been examined to determinetheir utility in the delivery of drugs to the skin, in particularsystems comprising non-ionic surfactant and cholesterol. Non-ionicliposomal formulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporin-A into different layers ofthe skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4, 6, 466).

[0104] Liposomes also include “sterically stabilized” liposomes, a termwhich, as used herein, refers to liposomes comprising one or morespecialized lipids that, when incorporated into liposomes, result inenhanced circulation lifetimes relative to liposomes lacking suchspecialized lipids. Examples of sterically stabilized liposomes arethose in which part of the vesicle-forming lipid portion of the liposome(A) comprises one or more glycolipids, such as monosialogangliosideG_(M1), or (B) is derivatized with one or more hydrophilic polymers,such as a polyethylene glycol (PEG) moiety. While not wishing to bebound by any particular theory, it is thought in the art that, at leastfor sterically stabilized liposomes containing gangliosides,sphingomyelin, or PEG-derivatized lipids, the enhanced circulationhalf-life of these sterically stabilized liposomes derives from areduced uptake into cells of the reticuloendothelial system (RES) (Allenet al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993,53, 3765).

[0105] Various liposomes comprising one or more glycolipids are known inthe art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside G_(M1), galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Limet al.).

[0106] Many liposomes comprising lipids derivatized with one or morehydrophilic polymers, and methods of preparation thereof, are known inthe art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778)described liposomes comprising a nonionic detergent, 2C₁₂15G, thatcontains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) notedthat hydrophilic coating of polystyrene particles with polymeric glycolsresults in significantly enhanced blood half-lives. Syntheticphospholipids modified by the attachment of carboxylic groups ofpolyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos.4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235)described experiments demonstrating that liposomes comprisingphosphatidylethanolamine (PE) derivatized with PEG or PEG stearate havesignificant increases in blood circulation half-lives. Blume et al.(Biochimica et Biophysica Acta, 1990, 1029, 91) extended suchobservations to other PEG-derivatized phospholipids, e.g., DSPE-PEG,formed from the combination of distearoylphosphatidylethanolamine (DSPE)and PEG. Liposomes having covalently bound PEG moieties on theirexternal surface are described in European Patent No. EP 0 445 131 B1and WO 90/04384 to Fisher. Liposome compositions containing 1-20 molepercent of PE derivatized with PEG, and methods of use thereof, aredescribed by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) andMartin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496813 B1). Liposomes comprising a number of other lipid-polymer conjugatesare disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martinet al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprisingPEG-modified ceramide lipids are described in WO 96/10391 (Choi et al.).U.S. Pat. Nos. 5,540,935 (Miyazaki et al.) and 5,556,948 (Tagawa et al.)describe PEG-containing liposomes that can be further derivatized withfunctional moieties on their surfaces.

[0107] A limited number of liposomes comprising nucleic acids are knownin the art. WO 96/40062 to Thierry et al. discloses methods forencapsulating high molecular weight nucleic acids in liposomes. U.S.Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomesand asserts that the contents of such liposomes may include an antisenseRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methodsof encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Loveet al. discloses liposomes comprising antisense oligonucleotidestargeted to the raf gene.

[0108] Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes may be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g. they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

[0109] Surfactants find wide application in formulations such asemulsions (including microemulsions) and liposomes. The most common wayof classifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988,p. 285).

[0110] If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

[0111] If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

[0112] If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

[0113] If the surfactant molecule has the ability to carry either apositive or negative charge, the surfactant is classified as amphoteric.Amphoteric surfactants include acrylic acid derivatives, substitutedalkylamides, N-alkylbetaines and phosphatides.

[0114] The use of surfactants in drug products, formulations and inemulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms,Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

[0115] Penetration Enhancers

[0116] In one embodiment, the present invention employs variouspenetration enhancers to effect the efficient delivery of nucleic acids,particularly oligonucleotides, to the skin of animals. Most drugs arepresent in solution in both ionized and nonionized forms. However,usually only lipid soluble or lipophilic drugs readily cross cellmembranes. It has been discovered that even non-lipophilic drugs maycross cell membranes if the membrane to be crossed is treated with apenetration enhancer. In addition to aiding the diffusion ofnon-lipophilic drugs across cell membranes, penetration enhancers alsoenhance the permeability of lipophilic drugs.

[0117] Penetration enhancers may be classified as belonging to one offive broad categories, i.e., surfactants, fatty acids, bile salts,chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92). Eachof the above mentioned classes of penetration enhancers are describedbelow in greater detail.

[0118] Surfactants:

[0119] In connection with the present invention, surfactants (or“surface-active agents”) are chemical entities which, when dissolved inan aqueous solution, reduce the surface tension of the solution or theinterfacial tension between the aqueous solution and another liquid,with the result that absorption of oligonucleotides through the mucosais enhanced. In addition to bile salts and fatty acids, thesepenetration enhancers include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991,p.92); and perfluorochemical emulsions, such as FC-43. Takahashi et al.,J. Pharm. Pharmacol., 1988, 40, 252).

[0120] Fatty Acids:

[0121] Various fatty acids and their derivatives which act aspenetration enhancers include, for example, oleic acid, lauric acid,capric acid (n-decanoic acid), myristic acid, palmitic acid, stearicacid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C₁₋₁₀ alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92;Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

[0122] Bile Salts:

[0123] The physiological role of bile includes the facilitation ofdispersion and absorption of lipids and fat-soluble vitamins (Brunton,Chapter 38 in: Goodman & Gilman's The Pharmacological Basis ofTherapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996,pp. 934-935). Various natural bile salts, and their syntheticderivatives, act as penetration enhancers. Thus the term “bile salts”includes any of the naturally occurring components of bile as well asany of their synthetic derivatives. The bile salts of the inventioninclude, for example, cholic acid (or its pharmaceutically acceptablesodium salt, sodium cholate), dehydrocholic acid (sodiumdehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid(sodium glucholate), glycholic acid (sodium glycocholate),glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid(sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate),chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid(UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodiumglycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee etal., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18thEd., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages782-783; Muranishi, Critical Reviews in Therapeutic Drug CarrierSystems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992,263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

[0124] Chelating Agents:

[0125] Chelating agents, as used in connection with the presentinvention, can be defined as compounds that remove metallic ions fromsolution by forming complexes therewith, with the result that absorptionof oligonucleotides through the mucosa is enhanced. With regards totheir use as penetration enhancers in the present invention, chelatingagents have the added advantage of also serving as DNase inhibitors, asmost characterized DNA nucleases require a divalent metal ion forcatalysis and are thus inhibited by chelating agents (Jarrett, J.Chromatogr., 1993, 618, 315-339). Chelating agents of the inventioninclude but are not limited to disodium ethylenediaminetetraacetate(EDTA), citric acid, salicylates (e.g., sodium salicylate,5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen,laureth-9 and N-amino acyl derivatives of beta-diketones (enamines) (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems,1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

[0126] Non-Chelating Non-Surfactants:

[0127] As used herein, non-chelating non-surfactant penetrationenhancing compounds can be defined as compounds that demonstrateinsignificant activity as chelating agents or as surfactants but thatnonetheless enhance absorption of oligonucleotides through thealimentary mucosa (Muranishi, Critical Reviews in Therapeutic DrugCarrier Systems, 1990, 7, 1-33). This class of penetration enhancersinclude, for example, unsaturated cyclic ureas, 1-alkyl- and1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, page 92); and non-steroidalanti-inflammatory agents such as diclofenac sodium, indomethacin andphenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39,621-626).

[0128] Agents that enhance uptake of oligonucleotides at the cellularlevel may also be added to the pharmaceutical and other compositions ofthe present invention. For example, cationic lipids, such as lipofectin(Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives,and polycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), are also known to enhance the cellular uptakeof oligonucleotides.

[0129] Other agents may be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

[0130] Carriers

[0131] Certain compositions of the present invention also incorporatecarrier compounds in the formulation. As used herein, “carrier compound”or “carrier” can refer to a nucleic acid, or analog thereof, which isinert (i.e., does not possess biological activity per se) but isrecognized as a nucleic acid by in vivo processes that reduce thebioavailability of a nucleic acid having biological activity by, forexample, degrading the biologically active nucleic acid or promoting itsremoval from circulation. The coadministration of a nucleic acid and acarrier compound, typically with an excess of the latter substance, canresult in a substantial reduction of the amount of nucleic acidrecovered in the liver, kidney or other extracirculatory reservoirs,presumably due to competition between the carrier compound and thenucleic acid for a common receptor. For example, the recovery of apartially phosphorothioate oligonucleotide in hepatic tissue can bereduced when it is coadministered with polyinosinic acid, dextransulfate, polycytidic acid or4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al.,Antisense Res. Dev., 1995, 5, 115-121; Takakura et al., Antisense &Nucl. Acid Drug Dev., 1996, 6, 177-183).

[0132] Excipients

[0133] In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient may be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc.) .

[0134] Pharmaceutically acceptable organic or inorganic excipientsuitable for non-parenteral administration which do not deleteriouslyreact with nucleic acids can also be used to formulate the compositionsof the present invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

[0135] Formulations for topical administration of nucleic acids mayinclude sterile and non-sterile aqueous solutions, non-aqueous solutionsin common solvents such as alcohols, or solutions of the nucleic acidsin liquid or solid oil bases. The solutions may also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

[0136] Suitable pharmaceutically acceptable excipients include, but arenot limited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

[0137] Other Components

[0138] The compositions of the present invention may additionallycontain other adjunct components conventionally found in pharmaceuticalcompositions, at their art-established usage levels. Thus, for example,the compositions may contain additional, compatible,pharmaceutically-active materials such as, for example, antipruritics,astringents, local anesthetics or anti-inflammatory agents, or maycontain additional materials useful in physically formulating variousdosage forms of the compositions of the present invention, such as dyes,flavoring agents, preservatives, antioxidants, opacifiers, thickeningagents and stabilizers. However, such materials, when added, should notunduly interfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

[0139] Aqueous suspensions may contain substances which increase theviscosity of the suspension including, for example, sodiumcarboxymethylcellulose, sorbitol and/or dextran. The suspension may alsocontain stabilizers.

[0140] Certain embodiments of the invention provide pharmaceuticalcompositions containing (a) one or more antisense compounds and (b) oneor more other chemotherapeutic agents which function by a non-antisensemechanism. Examples of such chemotherapeutic agents include but are notlimited to daunorubicin, daunomycin, dactinomycin, doxorubicin,epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide,cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C,actinomycin D, mithramycin, prednisone, hydroxyprogesterone,testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine,pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil,methylcyclohexylnitrosurea, nitrogen mustards, melphalan,cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine,5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol(DES). See, generally, The Merck Manual of Diagnosis and Therapy, 15thEd. 1987, pp. 1206-1228, Berkow et al., eds., Rahway, N.J. When usedwith the compounds of the invention, such chemotherapeutic agents may beused individually (e.g., 5-FU and oligonucleotide), sequentially (e.g.,5-FU and oligonucleotide for a period of time followed by MTX andoligonucleotide), or in combination with one or more other suchchemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU,radiotherapy and oligonucleotide). Anti-inflammatory drugs, includingbut not limited to nonsteroidal anti-inflammatory drugs andcorticosteroids, and antiviral drugs, including but not limited toribivirin, vidarabine, acyclovir and ganciclovir, may also be combinedin compositions of the invention. See, generally, The Merck Manual ofDiagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway,N.J., pages 2499-2506 and 46-49, respectively). Other non-antisensechemotherapeutic agents are also within the scope of this invention. Twoor more combined compounds may be used together or sequentially.

[0141] In another related embodiment, compositions of the invention maycontain one or more antisense compounds, particularly oligonucleotides,targeted to a first nucleic acid and one or more additional antisensecompounds targeted to a second nucleic acid target. Numerous examples ofantisense compounds are known in the art. Two or more combined compoundsmay be used together or sequentially.

[0142] The formulation of therapeutic compositions and their subsequentadministration is believed to be within the skill of those in the art.Dosing is dependent on severity and responsiveness of the disease stateto be treated, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient.Persons of ordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual oligonucleotides, and cangenerally be estimated based on EC₅₀s found to be effective in in vitroand in vivo animal models. In general, dosage is from 0.01 ug to 100 gper kg of body weight, and may be given once or more daily, weekly,monthly or yearly, or even once every 2 to 20 years. Persons of ordinaryskill in the art can easily estimate repetition rates for dosing basedon measured residence times and concentrations of the drug in bodilyfluids or tissues. Following successful treatment, it may be desirableto have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 ug to 100 g per kgof body weight, once or more daily, to once every 20 years.

[0143] While the present invention has been described with specificityin accordance with certain of its preferred embodiments, the followingexamples serve only to illustrate the invention and are not intended tolimit the same.

EXAMPLES Example 1 Nucleoside Phosphoramidites for OligonucleotideSynthesis Deoxy and 2′-alkoxy Amidites

[0144] 2′-Deoxy and 2′-methoxy beta-cyanoethyldiisopropylphosphoramidites were purchased from commercial sources (e.g. Chemgenes,Needham, Mass. or Glen Research, Inc. Sterling, Va.). Other 2′-O-alkoxysubstituted nucleoside amidites are prepared as described in U.S. Pat.No. 5,506,351, herein incorporated by reference. For oligonucleotidessynthesized using 2′-alkoxy amidites, optimized synthesis cycles weredeveloped that incorporate multiple steps coupling longer wait timesrelative to standard synthesis cycles.

[0145] The following abbreviations are used in the text: thin layerchromatography (TLC), melting point (MP), high pressure liquidchromatography (HPLC), Nuclear Magnetic Resonance (NMR), argon (Ar),methanol (MeOH), dichloromethane (CH₂Cl₂), triethylamine (TEA), dimethylformamide (DMF), ethyl acetate (EtOAc), dimethyl sulfoxide (DMSO),tetrahydrofuran (THF).

[0146] Oligonucleotides containing 5-methyl-2′-deoxycytidine (5-Me-dC)nucleotides were synthesized according to published methods (Sanghvi,et. al., Nucleic Acids Research, 1993, 21, 3197-3203) using commerciallyavailable phosphoramidites (Glen Research, Sterling, Va. or ChemGenes,Needham, Mass.) or prepared as follows:

[0147] Preparation of 5′-O-Dimethoxytrityl-thymidine Intermediate for5-methyl dC Amidite

[0148] To a 50 L glass reactor equipped with air stirrer and Ar gas linewas added thymidine (1.00 kg, 4.13 mol) in anhydrous pyridine (6 L) atambient temperature. Dimethoxytrityl (DMT) chloride (1.47 kg, 4.34 mol,1.05 eq) was added as a solid in four portions over 1 h. After 30 min,TLC indicated approx. 95% product, 2% thymidine, 5% DMT reagent andby-products and 2% 3′,5′-bis DMT product (R_(f) in EtOAc 0.45, 0.05,0.98, 0.95 respectively). Saturated sodium bicarbonate (4 L) and CH₂Cl₂were added with stirring (pH of the aqueous layer 7.5). An additional 18L of water was added, the mixture was stirred, the phases wereseparated, and the organic layer was transferred to a second 50 Lvessel. The aqueous layer was extracted with additional CH₂Cl₂ (2×2 L).The combined organic layer was washed with water (10 L) and thenconcentrated in a rotary evaporator to approx. 3.6 kg total weight. Thiswas redissolved in CH₂Cl₂ (3.5 L), added to the reactor followed bywater (6 L) and hexanes (13 L). The mixture was vigorously stirred andseeded to give a fine white suspended solid starting at the interface.After stirring for 1 h, the suspension was removed by suction through a½″ diameter teflon tube into a 20 L suction flask, poured onto a 25 cmCoors Buchner funnel, washed with water (2×3 L) and a mixture of hexanes—CH₂Cl₂ (4:1, 2×3 L) and allowed to air dry overnight in pans (1″ deep).This was further dried in a vacuum oven (75° C., 0.1 mm Hg, 48 h) to aconstant weight of 2072 g (93%) of a white solid, (mp 122-124° C.). TLCindicated a trace contamination of the bis DMT product. NMR spectroscopyalso indicated that 1-2 mole percent pyridine and about 5 mole percentof hexanes was still present.

[0149] Preparation of 5′-O-Dimethoxytrityl-2′-deoxy-5-methylcytidineIntermediate for 5-methyl-dC Amidite

[0150] To a 50 L Schott glass-lined steel reactor equipped with anelectric stirrer, reagent addition pump (connected to an additionfunnel), heating/cooling system, internal thermometer and an Ar gas linewas added 5′-O-dimethoxytrityl-thymidine (3.00 kg, 5.51 mol), anhydrousacetonitrile (25 L) and TEA (12.3 L, 88.4 mol, 16 eq). The mixture waschilled with stirring to −10° C. internal temperature (external −20°C.). Trimethylsilylchloride (2.1 L, 16.5 mol, 3.0 eq) was added over 30minutes while maintaining the internal temperature below −5° C.,followed by a wash of anhydrous acetonitrile (1 L). Note: the reactionis mildly exothermic and copious hydrochloric acid fumes form over thecourse of the addition. The reaction was allowed to warm to 0° C. andthe reaction progress was confirmed by TLC (EtOAc-hexanes 4:1; R_(f) 0.43 to 0.84 of starting material and silyl product, respectively). Uponcompletion, triazole (3.05 kg, 44 mol, 8.0 eq) was added the reactionwas cooled to −20° C. internal temperature (external −30° C.).Phosphorous oxychloride (1035 mL, 11.1 mol, 2.01 eq) was added over 60min so as to maintain the temperature between −20° C. and −10° C. duringthe strongly exothermic process, followed by a wash of anhydrousacetonitrile (1 L). The reaction was warmed to 0° C. and stirred for 1h. TLC indicated a complete conversion to the triazole product (R_(f)0.83 to 0.34 with the product spot glowing in long wavelength UV light).The reaction mixture was a peach-colored thick suspension, which turneddarker red upon warming without apparent decomposition. The reaction wascooled to −15° C. internal temperature and water (5 L) was slowly addedat a rate to maintain the temperature below +10° C. in order to quenchthe reaction and to form a homogenous solution. (Caution: this reactionis initially very strongly exothermic). Approximately one-half of thereaction volume (22 L) was transferred by air pump to another vessel,diluted with EtOAc (12 L) and extracted with water (2×8 L). The combinedwater layers were back-extracted with EtOAc (6 L). The water layer wasdiscarded and the organic layers were concentrated in a 20 L rotaryevaporator to an oily foam. The foam was coevaporated with anhydrousacetonitrile (4 L) to remove EtOAc. (note: dioxane may be used insteadof anhydrous acetonitrile if dried to a hard foam). The second half ofthe reaction was treated in the same way. Each residue was dissolved indioxane (3 L) and concentrated ammonium hydroxide (750 mL) was added. Ahomogenous solution formed in a few minutes and the reaction was allowedto stand overnight (although the reaction is complete within 1 h).

[0151] TLC indicated a complete reaction (product R_(f) 0.35 inEtOAc-MeOH 4:1). The reaction solution was concentrated on a rotaryevaporator to a dense foam. Each foam was slowly redissolved in warmEtOAc (4 L; 50° C.), combined in a 50 L glass reactor vessel, andextracted with water (2×4L) to remove the triazole by-product. The waterwas back-extracted with EtOAc (2 L). The organic layers were combinedand concentrated to about 8 kg total weight, cooled to 0° C. and seededwith crystalline product. After 24 hours, the first crop was collectedon a 25 cm Coors Buchner funnel and washed repeatedly with EtOAc (3×3L)until a white powder was left and then washed with ethyl ether (2×3L).The solid was put in pans (1″ deep) and allowed to air dry overnight.The filtrate was concentrated to an oil, then redissolved in EtOAc (2L), cooled and seeded as before. The second crop was collected andwashed as before (with proportional solvents) and the filtrate was firstextracted with water (2×1L) and then concentrated to an oil. The residuewas dissolved in EtOAc (1 L) and yielded a third crop which was treatedas above except that more washing was required to remove a yellow oilylayer.

[0152] After air-drying, the three crops were dried in a vacuum oven(50° C., 0.1 mm Hg, 24 h) to a constant weight (1750, 600 and 200 g,respectively) and combined to afford 2550 g (85%) of a white crystallineproduct (MP 215-217° C.) when TLC and NMR spectroscopy indicated purity.The mother liquor still contained mostly product (as determined by TLC)and a small amount of triazole (as determined by NMR spectroscopy), bisDMT product and unidentified minor impurities. If desired, the motherliquor can be purified by silica gel chromatography using a gradient ofMeOH (0-25%) in EtOAc to further increase the yield.

[0153] Preparation of5′-O-Dimethoxytrityl-2′-deoxy-N4-benzoyl-5-methylcytidine PenultimateIntermediate for 5-methyl dC Amidite

[0154] Crystalline 5′-O-dimethoxytrityl-5-methyl-2′-deoxycytidine (2000g, 3.68 mol) was dissolved in anhydrous DMF (6.0 kg) at ambienttemperature in a 50 L glass reactor vessel equipped with an air stirrerand argon line. Benzoic anhydride (Chem Impex not Aldrich, 874 g, 3.86mol, 1.05 eq) was added and the reaction was stirred at ambienttemperature for 8 h. TLC (CH₂Cl₂-EtOAc; CH₂Cl₂-EtOAc 4:1; R_(f) 0.25)indicated approx. 92% complete reaction. An additional amount of benzoicanhydride (44 g, 0.19 mol) was added. After a total of 18 h, TLCindicated approx. 96% reaction completion. The solution was diluted withEtOAc (20 L), TEA (1020 mL, 7.36 mol, ca 2.0 eq) was added withstirring, and the mixture was extracted with water (15 L, then 2×10 L).The aqueous layer was removed (no back-extraction was needed) and theorganic layer was concentrated in 2×20 L rotary evaporator flasks untila foam began to form. The residues were coevaporated with acetonitrile(1.5 L each) and dried (0.1 mm Hg, 25° C., 24 h) to 2520 g of a densefoam. High pressure liquid chromatography (HPLC) revealed acontamination of 6.3% of N4, 3′-O-dibenzoyl product, but very littleother impurities.

[0155] The product was purified by Biotage column chromatography (5 kgBiotage) prepared with 65:35:1 hexanes-EtOAc-TEA (4L). The crude product(800 g),dissolved in CH₂Cl₂ (2 L), was applied to the column. The columnwas washed with the 65:35:1 solvent mixture (20 kg), then 20:80:1solvent mixture (10 kg), then 99:1 EtOAc:TEA (17 kg). The fractionscontaining the product were collected, and any fractions containing theproduct and impurities were retained to be resubjected to columnchromatography. The column was re-equilibrated with the original 65:35:1solvent mixture (17 kg). A second batch of crude product (840 g) wasapplied to the column as before. The column was washed with thefollowing solvent gradients: 65:35:1 (9 kg), 55:45:1 (20 kg), 20:80:1(10 kg), and 99:1 EtOAc:TEA(15 kg). The column was reequilibrated asabove, and a third batch of the crude product (850 g) plus impurefractions recycled from the two previous columns (28 g) was purifiedfollowing the procedure for the second batch. The fractions containingpure product combined and concentrated on a 20L rotary evaporator,co-evaporated with acetontirile (3 L) and dried (0.1 mm Hg, 48 h, 25°C.) to a constant weight of 2023 g (85%) of white foam and 20 g ofslightly contaminated product from the third run. HPLC indicated apurity of 99.8% with the balance as the diBenzoyl product.

[0156][5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(5-methyl dC amidite)

[0157]5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N⁴-benzoyl-5-methylcytidine(998 g, 1.5 mol) was dissolved in anhydrous DMF (2 L). The solution wasco-evaporated with toluene (300 ml) at 50° C. under reduced pressure,then cooled to room temperature and 2-cyanoethyltetraisopropylphosphorodiamidite (680 g, 2.26 mol) and tetrazole (52.5g, 0.75 mol) were added. The mixture was shaken until all tetrazole wasdissolved, N-methylimidazole (15 ml) was added and the mixture was leftat room temperature for 5 hours. TEA (300 ml) was added, the mixture wasdiluted with DMF (2.5 L) and water (600 ml), and extracted with hexane(3×3 L). The mixture was diluted with water (1.2 L) and extracted with amixture of toluene (7.5 L) and hexane (6 L). The two layers wereseparated, the upper layer was washed with DMF-water (7:3 v/v, 3×2 L)and water (3×2 L), and the phases were separated. The organic layer wasdried (Na₂SO₄), filtered and rotary evaporated. The residue wasco-evaporated with acetonitrile (2×2 L) under reduced pressure and driedto a constant weight (25° C., 0.1 mm Hg, 40 h) to afford 1250 g anoff-white foam solid (96%).

[0158] 2′-Fluoro Amidites

[0159] 2′-Fluorodeoxyadenosine Amidites

[0160] 2′-fluoro oligonucleotides were synthesized as describedpreviously [Kawasaki, et. al., J. Med. Chem., 1993, 36, 831-841] andU.S. Pat. No. 5,670,633, herein incorporated by reference. Thepreparation of 2′-fluoropyrimidines containing a 5-methyl substitutionare described in U.S. Pat. No. 5,861,493. Briefly, the protectednucleoside N6-benzoyl-2′-deoxy-2′-fluoroadenosine was synthesizedutilizing commercially available 9-beta-D-arabinofuranosyladenine asstarting material and whereby the 2′-alpha-fluoro atom is introduced bya S_(N)2-displacement of a 2′-beta-triflate group. ThusN6-benzoyl-9-beta-D-arabinofuranosyladenine was selectively protected inmoderate yield as the 3′,5′-ditetrahydropyranyl (THP) intermediate.Deprotection of the THP and N6-benzoyl groups was accomplished usingstandard methodologies to obtain the 5′-dimethoxytrityl-(DMT) and5′-DMT-3′-phosphoramidite intermediates.

[0161] 2′-Fluorodeoxyguanosine

[0162] The synthesis of 2′-deoxy-2′-fluoroguanosine was accomplishedusing tetraisopropyldisiloxanyl (TPDS) protected9-beta-D-arabinofuranosylguanine as starting material, and conversion tothe intermediate isobutyryl-arabinofuranosylguanosine. Alternatively,isobutyryl-arabinofuranosylguanosine was prepared as described by Rosset al., (Nucleosides & Nucleosides, 16, 1645, 1997). Deprotection of theTPDS group was followed by protection of the hydroxyl group with THP togive isobutyryl di-THP protected arabinofuranosylguanine. SelectiveO-deacylation and triflation was followed by treatment of the crudeproduct with fluoride, then deprotection of the THP groups. Standardmethodologies were used to obtain the 5′-DMT- and5′-DMT-3′-phosphoramidites.

[0163] 2′-Fluorouridine

[0164] Synthesis of 2′-deoxy-2′-fluorouridine was accomplished by themodification of a literature procedure in which2,2′-anhydro-1-beta-D-arabinofuranosyluracil was treated with 70%hydrogen fluoride-pyridine. Standard procedures were used to obtain the5′-DMT and 5′-DMT-3′phosphoramidites.

[0165] 2′-Fluorodeoxycytidine

[0166] 2′-deoxy-2′-fluorocytidine was synthesized via amination of2′-deoxy-2′-fluorouridine, followed by selective protection to giveN4-benzoyl-2′-deoxy-2′-fluorocytidine. Standard procedures were used toobtain the 5′-DMT and 5′-DMT-3′phosphoramidites.

[0167] 2′-O-(2-Methoxyethyl) Modified Amidites

[0168] 2′-O-Methoxyethyl-substituted nucleoside amidites (otherwiseknown as MOE amidites) are prepared as follows, or alternatively, as perthe methods of Martin, P., (Helvetica Chimica Acta, 1995, 78, 486-504).

[0169] Preparation of 2′-O-(2-methoxyethyl)-5-methyluridine Intermediate

[0170] 2,2′-Anhydro-5-methyl-uridine (2000 g, 8.32 mol),tris(2-methoxyethyl)borate (2504 g, 10.60 mol), sodium bicarbonate (60g, 0.70 mol) and anhydrous 2-methoxyethanol (5 L) were combined in a 12L three necked flask and heated to 130° C. (internal temp) atatmospheric pressure, under an argon atmosphere with stirring for 21 h.TLC indicated a complete reaction. The solvent was removed under reducedpressure until a sticky gum formed (50-85° C. bath temp and 100-11 mmHg) and the residue was redissolved in water (3 L) and heated to boilingfor 30 min in order the hydrolyze the borate esters. The water wasremoved under reduced pressure until a foam began to form and then theprocess was repeated. HPLC indicated about 77% product, 15% dimer (5′ ofproduct attached to 2′ of starting material) and unknown derivatives,and the balance was a single unresolved early eluting peak.

[0171] The gum was redissolved in brine (3 L), and the flask was rinsedwith additional brine (3 L). The combined aqueous solutions wereextracted with chloroform (20 L) in a heavier-than continuous extractorfor 70 h. The chloroform layer was concentrated by rotary evaporation ina 20 L flask to a sticky foam (2400 g). This was coevaporated with MeOH(400 mL) and EtOAc (8 L) at 75° C. and 0.65 atm until the foam dissolvedat which point the vacuum was lowered to about 0.5 atm. After 2.5 L ofdistillate was collected a precipitate began to form and the flask wasremoved from the rotary evaporator and stirred until the suspensionreached ambient temperature. EtOAc (2 L) was added and the slurry wasfiltered on a 25 cm table top Buchner funnel and the product was washedwith EtOAc (3×2 L). The bright white solid was air dried in pans for 24h then further dried in a vacuum oven (50° C., 0.1 mm Hg, 24 h) toafford 1649 g of a white crystalline solid (mp 115.5-116.5° C.).

[0172] The brine layer in the 20 L continuous extractor was furtherextracted for 72 h with recycled chloroform. The chloroform wasconcentrated to 120 g of oil and this was combined with the motherliquor from the above filtration (225 g), dissolved in brine (250 mL)and extracted once with chloroform (250 mL). The brine solution wascontinuously extracted and the product was crystallized as describedabove to afford an additional 178 g of crystalline product containingabout 2% of thymine. The combined yield was 1827 g (69.4%). HPLCindicated about 99.5% purity with the balance being the dimer.

[0173] Preparation of 5′-O-DMT-2′-O-(2-methoxyethyl)-5-methyluridinePenultimate Intermediate

[0174] In a 50 L glass-lined steel reactor,2′-O-(2-methoxyethyl)-5-methyl-uridine (MOE-T, 1500 g, 4.738 mol),lutidine (1015 g, 9.476 mol) were dissolved in anhydrous acetonitrile(15 L). The solution was stirred rapidly and chilled to −10° C.(internal temperature). Dimethoxytriphenylmethyl chloride (1765.7 g,5.21 mol) was added as a solid in one portion. The reaction was allowedto warm to −2° C. over 1 h. (Note: The reaction was monitored closely byTLC (EtOAc) to determine when to stop the reaction so as to not generatethe undesired bis-DMT substituted side product). The reaction wasallowed to warm from −2 to 3° C. over 25 min. then quenched by addingMeOH (300 mL) followed after 10 min by toluene (16 L) and water (16 L).The solution was transferred to a clear 50 L vessel with a bottomoutlet, vigorously stirred for 1 minute, and the layers separated. Theaqueous layer was removed and the organic layer was washed successivelywith 10% aqueous citric acid (8 L) and water (12 L). The product wasthen extracted into the aqueous phase by washing the toluene solutionwith aqueous sodium hydroxide (0.5N, 16 L and 8 L). The combined aqueouslayer was overlayed with toluene (12 L) and solid citric acid (8 moles,1270 g) was added with vigorous stirring to lower the pH of the aqueouslayer to 5.5 and extract the product into the toluene. The organic layerwas washed with water (10 L) and TLC of the organic layer indicated atrace of DMT-O-Me, bis DMT and dimer DMT.

[0175] The toluene solution was applied to a silica gel column (6 Lsintered glass funnel containing approx. 2 kg of silica gel slurriedwith toluene (2 L) and TEA (25 mL)) and the fractions were eluted withtoluene (12 L) and EtOAc (3×4 L) using vacuum applied to a filter flaskplaced below the column. The first EtOAc fraction containing both thedesired product and impurities were resubjected to column chromatographyas above. The clean fractions were combined, rotary evaporated to afoam, coevaporated with acetonitrile (6 L) and dried in a vacuum oven(0.1 mm Hg, 40 h, 40° C.) to afford 2850 g of a white crisp foam. NMRspectroscopy indicated a 0.25 mole % remainder of acetonitrile(calculates to be approx. 47 g) to give a true dry weight of 2803 g(96%). HPLC indicated that the product was 99.41% pure, with theremainder being 0.06 DMT-O-Me, 0.10 unknown, 0.44 bis DMT, and nodetectable dimer DMT or 3′-O-DMT.

[0176] Preparation of[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE T Amidite)

[0177]5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridine(1237 g, 2.0 mol) was dissolved in anhydrous DMF (2.5 L). The solutionwas co-evaporated with toluene (200 ml) at 50° C. under reducedpressure, then cooled to room temperature and 2-cyanoethyltetraisopropylphosphorodiamidite (900 g, 3.0 mol) and tetrazole (70 g,1.0 mol) were added. The mixture was shaken until all tetrazole wasdissolved, N-methylimidazole (20 ml) was added and the solution was leftat room temperature for 5 hours. TEA (300 ml) was added, the mixture wasdiluted with DMF (3.5 L) and water (600 ml) and extracted with hexane(3×3L). The mixture was diluted with water (1.6 L) and extracted withthe mixture of toluene (12 L) and hexanes (9 L). The upper layer waswashed with DMF-water (7:3 v/v, 3×3 L) and water (3×3 L). The organiclayer was dried (Na₂SO₄), filtered and evaporated. The residue wasco-evaporated with acetonitrile (2×2 L) under reduced pressure and driedin a vacuum oven (25° C., 0.1 mm Hg, 40 h) to afford 1526 g of anoff-white foamy solid (95%).

[0178] Preparation of5′-O-Dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methylcytidine Intermediate

[0179] To a 50 L Schott glass-lined steel reactor equipped with anelectric stirrer, reagent addition pump (connected to an additionfunnel), heating/cooling system, internal thermometer and argon gas linewas added 5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methyl-uridine(2.616 kg, 4.23 mol, purified by base extraction only and no scrubcolumn), anhydrous acetonitrile (20 L), and TEA (9.5 L, 67.7 mol, 16eq). The mixture was chilled with stirring to −10° C. internaltemperature (external −20° C.). Trimethylsilylchloride (1.60 L, 12.7mol, 3.0 eq) was added over 30 min. while maintaining the internaltemperature below −5° C., followed by a wash of anhydrous acetonitrile(1 L). (Note: the reaction is mildly exothermic and copious hydrochloricacid fumes form over the course of the addition). The reaction wasallowed to warm to 0° C. and the reaction progress was confirmed by TLC(EtOAc, R_(f) 0.68 and 0.87 for starting material and silyl product,respectively). Upon completion, triazole (2.34 kg, 33.8 mol, 8.0 eq) wasadded the reaction was cooled to −20° C. internal temperature (external−30° C.). Phosphorous oxychloride (793 mL, 8.51 mol, 2.01 eq) was addedslowly over 60 min so as to maintain the temperature between −20° C. and−10° C. (note: strongly exothermic), followed by a wash of anhydrousacetonitrile (1 L). The reaction was warmed to 0° C. and stirred for 1h, at which point it was an off-white thick suspension. TLC indicated acomplete conversion to the triazole product (EtOAc, R_(f) 0.87 to 0.75with the product spot glowing in long wavelength UV light). The reactionwas cooled to −15° C. and water (5 L) was slowly added at a rate tomaintain the temperature below +10° C. in order to quench the reactionand to form a homogenous solution. (Caution: this reaction is initiallyvery strongly exothermic). Approximately one-half of the reaction volume(22 L) was transferred by air pump to another vessel, diluted with EtOAc(12 L) and extracted with water (2×8 L). The second half of the reactionwas treated in the same way. The combined aqueous layers wereback-extracted with EtOAc (8 L) The organic layers were combined andconcentrated in a 20 L rotary evaporator to an oily foam. The foam wascoevaporated with anhydrous acetonitrile (4 L) to remove EtOAc. (note:dioxane may be used instead of anhydrous acetonitrile if dried to a hardfoam). The residue was dissolved in dioxane (2 L) and concentratedammonium hydroxide (750 mL) was added. A homogenous solution formed in afew minutes and the reaction was allowed to stand overnight

[0180] TLC indicated a complete reaction (CH₂Cl₂-acetone-MeOH, 20:5:3,R_(f) 0.51). The reaction solution was concentrated on a rotaryevaporator to a dense foam and slowly redissolved in warm CH₂Cl₂ (4 L,40° C.) and transferred to a 20 L glass extraction vessel equipped witha air-powered stirrer. The organic layer was extracted with water (2×6L) to remove the triazole by-product. (Note: In the first extraction anemulsion formed which took about 2 h to resolve). The water layer wasback-extracted with CH₂Cl₂ (2×2 L), which in turn was washed with water(3 L). The combined organic layer was concentrated in 2×20 L flasks to agum and then recrystallized from EtOAc seeded with crystalline product.After sitting overnight, the first crop was collected on a 25 cm CoorsBuchner funnel and washed repeatedly with EtOAc until a whitefree-flowing powder was left (about 3×3 L). The filtrate wasconcentrated to an oil recrystallized from EtOAc, and collected asabove. The solid was air-dried in pans for 48 h, then further dried in avacuum oven (50° C., 0.1 mm Hg, 17 h) to afford 2248 g of a brightwhite, dense solid (86%). An HPLC analysis indicated both crops to be99.4% pure and NMR spectroscopy indicated only a faint trace of EtOAcremained.

[0181] Preparation of5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-N4-benzoyl-5-methyl-cytidinePenultimate Intermediate:

[0182] Crystalline5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methyl-cytidine (1000 g,1.62 mol) was suspended in anhydrous DMF (3 kg) at ambient temperatureand stirred under an Ar atmosphere. Benzoic anhydride (439.3 g, 1.94mol) was added in one portion. The solution clarified after 5 hours andwas stirred for 16 h. HPLC indicated 0.45% starting material remained(as well as 0.32% N4, 3′-O-bis Benzoyl). An additional amount of benzoicanhydride (6.0 g, 0.0265 mol) was added and after 17 h, HPLC indicatedno starting material was present. TEA (450 mL, 3.24 mol) and toluene (6L) were added with stirring for 1 minute. The solution was washed withwater (4×4 L), and brine (2×4 L). The organic layer was partiallyevaporated on a 20 L rotary evaporator to remove 4 L of toluene andtraces of water. HPLC indicated that the bis benzoyl side product waspresent as a 6% impurity. The residue was diluted with toluene (7 L) andanhydrous DMSO (200 mL, 2.82 mol) and sodium hydride (60% in oil, 70 g,1.75 mol) was added in one portion with stirring at ambient temperatureover 1 h. The reaction was quenched by slowly adding then washing withaqueous citric acid (10%, 100 mL over 10 min, then 2×4 L), followed byaqueous sodium bicarbonate (2%, 2 L), water (2×4 L) and brine (4 L). Theorganic layer was concentrated on a 20 L rotary evaporator to about 2 Ltotal volume. The residue was purified by silica gel columnchromatography (6 L Buchner funnel containing 1.5 kg of silica gelwetted with a solution of EtOAc-hexanes-TEA (70:29:1)). The product waseluted with the same solvent (30 L) followed by straight EtOAc (6 L).The fractions containing the product were combined, concentrated on arotary evaporator to a foam and then dried in a vacuum oven (50° C., 0.2mm Hg, 8 h) to afford 1155 g of a crisp, white foam (98%). HPLCindicated a purity of >99.7%.

[0183] Preparation of[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE 5-Me-C Amidite)

[0184]5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methylcytidine(1082 g, 1.5 mol) was dissolved in anhydrous DMF (2 L) and co-evaporatedwith toluene (300 ml) at 50° C. under reduced pressure. The mixture wascooled to room temperature and 2-cyanoethyltetraisopropylphosphorodiamidite (680 g, 2.26 mol) and tetrazole (52.5g, 0.75 mol) were added. The mixture was shaken until all tetrazole wasdissolved, N-methylimidazole (30 ml) was added, and the mixture was leftat room temperature for 5 hours. TEA (300 ml) was added, the mixture wasdiluted with DMF (1 L) and water (400 ml) and extracted with hexane (3×3L). The mixture was diluted with water (1.2 L) and extracted with amixture of toluene (9 L) and hexanes (6 L). The two layers wereseparated and the upper layer was washed with DMF-water (60:40 v/v, 3×3L) and water (3×2 L). The organic layer was dried (Na₂SO₄), filtered andevaporated. The residue was co-evaporated with acetonitrile (2×2 L)under reduced pressure and dried in a vacuum oven (25° C., 0.1 mm Hg, 40h) to afford 1336 g of an off-white foam (97%).

[0185] Preparation of[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁶-benzoyladenosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE A Amdite)

[0186]5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁶-benzoyladenosine(purchased from Reliable Biopharmaceutical, St. Lois, Mo.), 1098 g, 1.5mol) was dissolved in anhydrous DMF (3 L) and co-evaporated with toluene(300 ml) at 50° C. The mixture was cooled to room temperature and2-cyanoethyl tetraisopropylphosphorodiamidite (680 g, 2.26 mol) andtetrazole (78.8 g, 1.24 mol) were added. The mixture was shaken untilall tetrazole was dissolved, N-methylimidazole (30 ml) was added, andmixture was left at room temperature for 5 hours. TEA (300 ml) wasadded, the mixture was diluted with DMF (1 L) and water (400 ml) andextracted with hexanes (3×3 L). The mixture was diluted with water (1.4L) and extracted with the mixture of toluene (9 L) and hexanes (6 L).The two layers were separated and the upper layer was washed withDMF-water (60:40, v/v, 3×3 L) and water (3×2 L). The organic layer wasdried (Na₂SO₄), filtered and evaporated to a sticky foam. The residuewas co-evaporated with acetonitrile (2.5 L) under reduced pressure anddried in a vacuum oven (25° C., 0.1 mm Hg, 40 h) to afford 1350 g of anoff-white foam solid (96%).

[0187] Prepartion of[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-isobutyrylguanosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE G Amidite)

[0188]5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-isobutyrlguanosine(purchased from Reliable Biopharmaceutical, St. Louis, Mo., 1426 g, 2.0mol) was dissolved in anhydrous DMF (2 L). The solution wasco-evaporated with toluene (200 ml) at 50° C., cooled to roomtemperature and 2-cyanoethyl tetraisopropylphosphorodiamidite (900 g,3.0 mol) and tetrazole (68 g, 0.97 mol) were added. The mixture wasshaken until all tetrazole was dissolved, N-methylimidazole (30 ml) wasadded, and the mixture was left at room temperature for 5 hours. TEA(300 ml) was added, the mixture was diluted with DMF (2 L) and water(600 ml) and extracted with hexanes (3×3 L). The mixture was dilutedwith water (2 L) and extracted with a mixture of toluene (10 L) andhexanes (5 L). The two layers were separated and the upper layer waswashed with DMF-water (60:40, v/v, 3×3 L). EtOAc (4 L) was added and thesolution was washed with water (3×4 L). The organic layer was dried(Na₂SO₄), filtered and evaporated to approx. 4 kg. Hexane (4 L) wasadded, the mixture was shaken for 10 min, and the supernatant liquid wasdecanted. The residue was co-evaporated with acetonitrile (2×2 L) underreduced pressure and dried in a vacuum oven (25° C., 0.1 mm Hg, 40 h) toafford 1660 g of an off-white foamy solid (91%).

[0189] 2′-O-(Aminooxyethyl) Nucleoside Amidites and2′-O-(dimethylaminooxyethyl) Nucleoside Amidites

[0190] 2′-(Dimethylaminooxyethoxy) Nucleoside Amidites

[0191] 2′-(Dimethylaminooxyethoxy) nucleoside amidites (also known inthe art as 2′-O-(dimethylaminooxyethyl) nucleoside amidites) areprepared as described in the following paragraphs. Adenosine, cytidineand guanosine nucleoside amidites are prepared similarly to thethymidine (5-methyluridine) except the exocyclic amines are protectedwith a benzoyl moiety in the case of adenosine and cytidine and withisobutyryl in the case of guanosine.

[0192] 5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine

[0193] O²-2′-anhydro-5-methyluridine (Pro. Bio. Sint., Varese, Italy,100.0 g, 0.416 mmol), dimethylaminopyridine (0.66 g, 0.013 eq, 0.0054mmol) were dissolved in dry pyridine (500 ml) at ambient temperatureunder an argon atmosphere and with mechanical stirring.tert-Butyldiphenylchlorosilane (125.8 g, 119.0 mL, 1.1 eq, 0.458 mmol)was added in one portion. The reaction was stirred for 16 h at ambienttemperature. TLC (R_(f) 0.22, EtOAc) indicated a complete reaction. Thesolution was concentrated under reduced pressure to a thick oil. Thiswas partitioned between CH₂Cl₂ (1 L) and saturated sodium bicarbonate(2×1 L) and brine (1 L). The organic layer was dried over sodiumsulfate, filtered, and concentrated under reduced pressure to a thickoil. The oil was dissolved in a 1:1 mixture of EtOAc and ethyl ether(600 mL) and cooling the solution to −10° C. afforded a whitecrystalline solid which was collected by filtration, washed with ethylether (3×200 mL) and dried (40° C., 1 mm Hg, 24 h) to afford 149 g ofwhite solid (74.8%). TLC and NMR spectroscopy were consistent with pureproduct.

[0194] 5′-O-tert-Butyldiphenylsilyl-2-O-(2-hydroxyethyl)-5-methyluridine

[0195] In the fume hood, ethylene glycol (350 mL, excess) was addedcautiously with manual stirring to a 2 L stainless steel pressurereactor containing borane in tetrahydrofuran (1.0 M, 2.0 eq, 622 mL).(Caution: evolves hydrogen gas).5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine (149 g, 0.311mol) and sodium bicarbonate (0.074 g, 0.003 eq) were added with manualstirring. The reactor was sealed and heated in an oil bath until aninternal temperature of 160° C. was reached and then maintained for 16 h(pressure <100 psig). The reaction vessel was cooled to ambienttemperature and opened. TLC (EtOAc, R_(f) 0.67 for desired product andR_(f) 0.82 for ara-T side product) indicated about 70% conversion to theproduct. The solution was concentrated under reduced pressure (10 to 1mm Hg) in a warm water bath (40-100° C.) with the more extremeconditions used to remove the ethylene glycol. (Alternatively, once theTHF has evaporated the solution can be diluted with water and theproduct extracted into EtOAc). The residue was purified by columnchromatography (2 kg silica gel, EtOAc-hexanes gradient 1:1 to 4:1). Theappropriate fractions were combined, evaporated and dried to afford 84 gof a white crisp foam (50%), contaminated starting material (17.4 g, 12%recovery) and pure reusable starting material (20 g, 13% recovery). TLCand NMR spectroscopy were consistent with 99% pure product.

[0196]2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine

[0197]5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine (20g,36.98mmol) was mixed with triphenylphosphine (11.63g, 44.36mmol) andN-hydroxyphthalimide (7.24g, 44.36mmol) and dried over P₂O₅ under highvacuum for two days at 40° C. The reaction mixture was flushed withargon and dissolved in dry THF (369.8 mL, Aldrich, sure seal bottle).Diethyl-azodicarboxylate (6.98 mL, 44.36 mmol) was added dropwise to thereaction mixture with the rate of addition maintained such that theresulting deep red coloration is just discharged before adding the nextdrop. The reaction mixture was stirred for 4 hrs., after which time TLC(EtOAc:hexane, 60:40) indicated that the reaction was complete. Thesolvent was evaporated in vacuuo and the residue purified by flashcolumn chromatography (eluted with 60:40 EtOAc:hexane), to yield2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine aswhite foam (21.819 g, 86%) upon rotary evaporation.

[0198]5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine

[0199]2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine(3.1 g, 4.5 mmol) was dissolved in dry CH₂Cl₂ (4.5 mL) andmethylhydrazine (300 mL, 4.64 mmol) was added dropwise at −10° C. to 0°C. After 1 h the mixture was filtered, the filtrate washed with ice coldCH₂Cl₂, and the combined organic phase was washed with water and brineand dried (anhydrous Na₂SO₄). The solution was filtered and evaporatedto afford 2′-O-(aminooxyethyl) thymidine, which was then dissolved inMeOH (67.5 mL). Formaldehyde (20% aqueous solution, w/w, 1.1 eq.) wasadded and the resulting mixture was stirred for 1 h. The solvent wasremoved under vacuum and the residue was purified by columnchromatography to yield5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridineas white foam (1.95 g, 78%) upon rotary evaporation.

[0200] 5′-O-tert-Butyldiphenylsilyl-2′-O-[N,Ndimethylaminooxyethyl]-5-methyluridine

[0201]5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine(1.77g, 3.12mmol) was dissolved in a solution of 1M pyridiniump-toluenesulfonate (PPTS) in dry MeOH (30.6 mL) and cooled to 10° C.under inert atmosphere. Sodium cyanoborohydride (0.39 g, 6.13 mmol) wasadded and the reaction mixture was stirred. After 10 minutes thereaction was warmed to room temperature and stirred for 2 h. while theprogress of the reaction was monitored by TLC (5% MeOH in CH₂Cl₂).Aqueous NaHCO₃ solution (5%, 10 mL) was added and the product wasextracted with EtOAc (2×20 mL). The organic phase was dried overanhydrous Na₂SO₄, filtered, and evaporated to dryness. This entireprocedure was repeated with the resulting residue, with the exceptionthat formaldehyde (20% w/w, 30 mL, 3.37 mol) was added upon dissolutionof the residue in the PPTS/MeOH solution. After the extraction andevaporation, the residue was purified by flash column chromatography and(eluted with 5% MeOH in CH₂Cl₂) to afford5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridineas a white foam (14.6 g, 80%) upon rotary evaporation.

[0202] 2′-O-(dimethylaminooxyethyl)-5-methyluridine

[0203] Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) was dissolvedin dry THF and TEA (1.67 mL, 12 mmol, dry, stored over KOH) and added to5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine(1.40 g, 2.4mmol). The reaction was stirred at room temperature for 24hrs and monitored by TLC (5% MeOH in CH₂Cl₂). The solvent was removedunder vacuum and the residue purified by flash column chromatography(eluted with 10% MeOH in CH₂Cl₂) to afford2′-O-(dimethylaminooxyethyl)-5-methyluridine (766 mg, 92.5%) upon rotaryevaporation of the solvent.

[0204] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine

[0205] 2′-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17 mmol)was dried over P₂O₅ under high vacuum overnight at 40° C., co-evaporatedwith anhydrous pyridine (20 mL), and dissolved in pyridine (11 mL) underargon atmosphere. 4-dimethylaminopyridine (26.5 mg, 2.60 mmol) and4,4′-dimethoxytrityl chloride (880 mg, 2.60 mmol) were added to thepyridine solution and the reaction mixture was stirred at roomtemperature until all of the starting material had reacted. Pyridine wasremoved under vacuum and the residue was purified by columnchromatography (eluted with 10% MeOH in CH₂Cl₂ containing a few drops ofpyridine) to yield5′-O-DMT-2′-O-(dimethylamino-oxyethyl)-5-methyluridine (1.13 g, 80%)upon rotary evaporation.

[0206]5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

[0207] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine (1.08 g,1.67 mmol) was co-evaporated with toluene (20 mL), N,N-diisopropylaminetetrazonide (0.29 g, 1.67 mmol) was added and the mixture was dried overP₂O₅ under high vacuum overnight at 40° C. This was dissolved inanhydrous acetonitrile (8.4 mL) and2-cyanoethyl-N,N,N¹,N¹-tetraisopropylphosphoramidite (2.12 mL, 6.08mmol) was added. The reaction mixture was stirred at ambient temperaturefor 4 h under inert atmosphere. The progress of the reaction wasmonitored by TLC (hexane:EtOAc 1:1). The solvent was evaporated, thenthe residue was dissolved in EtOAc (70 mL) and washed with 5% aqueousNaHCO₃ (40 mL) The EtOAc layer was dried over anhydrous Na₂SO₄,filtered, and concentrated. The residue obtained was purified by columnchromatography (EtOAc as eluent) to afford5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]as a foam (1.04 g, 74.9%) upon rotary evaporation.

[0208] 2′-(Aminooxyethoxy) Nucleoside Amidites

[0209] 2′-(Aminooxyethoxy) nucleoside amidites (also known in the art as2′-O-(aminooxyethyl) nucleoside amidites) are prepared as described inthe following paragraphs. Adenosine, cytidine and thymidine nucleosideamidites are prepared similarly.

[0210]N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

[0211] The 2′-O-aminooxyethyl guanosine analog may be obtained byselective 2′-O-alkylation of diaminopurine riboside. Multigramquantities of diaminopurine riboside may be purchased from Schering AG(Berlin) to provide 2′-O-(2-ethylacetyl) diaminopurine riboside alongwith a minor amount of the 3′-O-isomer. 2′-O-(2-ethylacetyl)diaminopurine riboside may be resolved and converted to2′-O-(2-ethylacetyl) guanosine by treatment with adenosine deaminase.(McGee, D. P. C., Cook, P. D., Guinosso, C. J., WO 94/02501 A1 940203.)Standard protection procedures should afford2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine and2-N-isobutyryl-6-O-diphenylcarbamoyl-2-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine which may be reduced to provide2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-hydroxyethyl)-5′-O-(4,4′-dimethoxytrityl)guanosine.As before the hydroxyl group may be displaced by N-hydroxyphthalimidevia a Mitsunobu reaction, and the protected nucleoside may bephosphitylated as usual to yield2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-([2-phthalmidoxy]ethyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite].

[0212] 2′-dimethylaminoethoxyethoxy (2′-DMAEOE) Nucleoside Amidites

[0213] 2′-dimethylaminoethoxyethoxy nucleoside amidites (also known inthe art as 2′-O-dimethylaminoethoxyethyl, i.e., 2′—O—CH₂—O—CH₂—N(CH₂)₂,or 2′-DMAEOE nucleoside amidites) are prepared as follows. Othernucleoside amidites are prepared similarly.

[0214] 2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl Uridine

[0215] 2[2-(Dimethylamino)ethoxy]ethanol (Aldrich, 6.66 g, 50 mmol) wasslowly added to a solution of borane in tetra-hydrofuran (1 M, 10 mL, 10mmol) with stirring in a 100 mL bomb. (Caution: Hydrogen gas evolves asthe solid dissolves). O²-,2′-anhydro-5-methyluridine (1.2 g, 5 mmol),and sodium bicarbonate (2.5 mg) were added and the bomb was sealed,placed in an oil bath and heated to 155° C. for 26 h. then cooled toroom temperature. The crude solution was concentrated, the residue wasdiluted with water (200 mL) and extracted with hexanes (200 mL). Theproduct was extracted from the aqueous layer with EtOAc (3×200 mL) andthe combined organic layers were washed once with water, dried overanhydrous sodium sulfate, filtered and concentrated. The residue waspurified by silica gel column chromatography (eluted with 5:100:2MeOH/CH₂Cl₂/TEA) as the eluent. The appropriate fractions were combinedand evaporated to afford the product as a white solid.

[0216] 5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyl Uridine

[0217] To 0.5 g (1.3 mmol) of2′-O-[2(2-N,N-dimethylamino-ethoxy)ethyl)]-5-methyl uridine in anhydrouspyridine (8 mL), was added TEA (0.36 mL) and dimethoxytrityl chloride(DMT-Cl, 0.87 g, 2 eq.) and the reaction was stirred for 1 h. Thereaction mixture was poured into water (200 mL) and extracted withCH₂Cl₂ (2×200 mL). The combined CH₂Cl₂ layers were washed with saturatedNaHCO₃ solution, followed by saturated NaCl solution, dried overanhydrous sodium sulfate, filtered and evaporated. The residue waspurified by silica gel column chromatography (eluted with 5:100:1MeOH/CH₂Cl₂/TEA) to afford the product.

[0218]5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methylUridine-3′-O-(cyanoethyl-N,N-diisopropyl)Phosphoramidite

[0219] Diisopropylaminotetrazolide (0.6 g) and2-cyanoethoxy-N,N-diisopropyl phosphoramidite (1.1 mL, 2 eq.) were addedto a solution of5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluridine(2.17 g, 3 mmol) dissolved in CH₂Cl₂ (20 mL) under an atmosphere ofargon. The reaction mixture was stirred overnight and the solventevaporated. The resulting residue was purified by silica gel columnchromatography with EtOAc as the eluent to afford the title compound.

Example 2 Oligonucleotide Synthesis

[0220] Unsubstituted and substituted phosphodiester (P═O)oligonucleotides are synthesized on an automated DNA synthesizer(Applied Biosystems model 394) using standard phosphoramidite chemistrywith oxidation by iodine.

[0221] Phosphorothioates (P═S) are synthesized similar to phosphodiesteroligonucleotides with the following exceptions: thiation was effected byutilizing a 10% w/v solution of 3H-1,2-benzodithiole-3-one 1,1-dioxidein acetonitrile for the oxidation of the phosphite linkages. Thethiation reaction step time was increased to 180 sec and preceded by thenormal capping step. After cleavage from the CPG column and deblockingin concentrated ammonium hydroxide at 55° C. (12-16 hr), theoligonucleotides were recovered by precipitating with >3 volumes ofethanol from a 1 M NH₄OAc solution. Phosphinate oligonucleotides areprepared as described in U.S. Pat. No. 5,508,270, herein incorporated byreference.

[0222] Alkyl phosphonate oligonucleotides are prepared as described inU.S. Pat. No. 4,469,863, herein incorporated by reference.

[0223] 3′-Deoxy-3′-methylene phosphonate oligonucleotides are preparedas described in U.S. Pat. Nos. 5,610,289 or 5,625,050, hereinincorporated by reference.

[0224] Phosphoramidite oligonucleotides are prepared as described inU.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporatedby reference.

[0225] Alkylphosphonothioate oligonucleotides are prepared as describedin published PCT applications PCT/US94/00902 and PCT/US93/06976(published as WO 94/17093 and WO 94/02499, respectively), hereinincorporated by reference.

[0226] 3′-Deoxy-3′-amino phosphoramidate oligonucleotides are preparedas described in U.S. Pat. No. 5,476,925, herein incorporated byreference.

[0227] Phosphotriester oligonucleotides are prepared as described inU.S. Pat. No. 5,023,243, herein incorporated by reference.

[0228] Borano phosphate oligonucleotides are prepared as described inU.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated byreference.

Example 3 Oligonucleoside Synthesis

[0229] Methylenemethylimino linked oligonucleosides, also identified asMMI linked oligonucleosides, methylenedimethyl-hydrazo linkedoligonucleosides, also identified as MDH linked oligonucleosides, andmethylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified as amide-4 linked oligonucleosides, aswell as mixed backbone compounds having, for instance, alternating MMIand P═O or P═S linkages are prepared as described in U.S. Pat. Nos.5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of whichare herein incorporated by reference.

[0230] Formacetal and thioformacetal linked oligonucleosides areprepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564, hereinincorporated by reference.

[0231] Ethylene oxide linked oligonucleosides are prepared as describedin U.S. Pat. No. 5,223,618, herein incorporated by reference.

Example 4 PNA Synthesis

[0232] Peptide nucleic acids (PNAs) are prepared in accordance with anyof the various procedures referred to in Peptide Nucleic Acids (PNA):Synthesis, Properties and Potential Applications, Bioorganic & MedicinalChemistry, 1996, 4, 5-23. They may also be prepared in accordance withU.S. Pat. Nos. 5,539,082, 5,700,922, and 5,719,262, herein incorporatedby reference.

Example 5 Synthesis of Chimeric Oligonucleotides

[0233] Chimeric oligonucleotides, oligonucleosides or mixedoligonucleotides/oligonucleosides of the invention can be of severaldifferent types. These include a first type wherein the “gap” segment oflinked nucleosides is positioned between 5′ and 3′ “wing” segments oflinked nucleosides and a second “open end” type wherein the “gap”segment is located at either the 3′ or the 5′ terminus of the oligomericcompound. Oligonucleotides of the first type are also known in the artas “gapmers” or gapped oligonucleotides. Oligonucleotides of the secondtype are also known in the art as “hemimers” or “wingmers”.

[0234] [2′-O-Me]--[2′-deoxy]--[2′-O-Me] Chimeric PhosphorothioateOligonucleotides

[0235] Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and2′-deoxy phosphorothioate oligonucleotide segments are synthesized usingan Applied Biosystems automated DNA synthesizer Model 394, as above.Oligonucleotides are synthesized using the automated synthesizer and2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′and 3′wings.The standard synthesis cycle is modified by incorporating coupling stepswith increased reaction times for the5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite. The fully protectedoligonucleotide is cleaved from the support and deprotected inconcentrated ammonia (NH₄OH) for 12-16 hr at 55° C. The deprotectedoligo is then recovered by an appropriate method (precipitation, columnchromatography, volume reduced in vacuo and analyzedspetrophotometrically for yield and for purity by capillaryelectrophoresis and by mass spectrometry.

[0236] [2′-O-(2-Methoxyethyl)]--[2′-deoxy]--[2′-O-(Methoxyethyl)]Chimeric Phosphorothioate Oligonucleotides

[0237] [2′-O-(2-methoxyethyl)]--[2′-deoxy]--[-2′-O-(methoxyethyl)]chimeric phosphorothioate oligonucleotides were prepared as per theprocedure above for the 2′-O-methyl chimeric oligonucleotide, with thesubstitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methylamidites.

[0238] [2′-O-(2-Methoxyethyl)Phosphodiester]--[2′-deoxyPhosphorothioate]--[2′-O-(2-Methoxyethyl) Phosphodiester] ChimericOligonucleotides

[0239] [2′-O-(2-methoxyethyl phosphodiester]--[2′-deoxyphosphorothioate]--[2′-O-(methoxyethyl) phosphodiester] chimericoligonucleotides are prepared as per the above procedure for the2′-O-methyl chimeric oligonucleotide with the substitution of2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidationwith iodine to generate the phosphodiester internucleotide linkageswithin the wing portions of the chimeric structures and sulfurizationutilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) togenerate the phosphorothioate internucleotide linkages for the centergap.

[0240] Other chimeric oligonucleotides, chimeric oligonucleosides andmixed chimeric oligonucleotides/oligonucleosides are synthesizedaccording to U.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 6 Oligonucleotide Isolation

[0241] After cleavage from the controlled pore glass solid support anddeblocking in concentrated ammonium hydroxide at 55° C. for 12-16 hours,the oligonucleotides or oligonucleosides are recovered by precipitationout of 1 M NH₄OAc with >3 volumes of ethanol. Synthesizedoligonucleotides were analyzed by electrospray mass spectroscopy(molecular weight determination) and by capillary gel electrophoresisand judged to be at least 70% full length material. The relative amountsof phosphorothioate and phosphodiester linkages obtained in thesynthesis was determined by the ratio of correct molecular weightrelative to the −16 amu product (+/−32+/−48). For some studiesoligonucleotides were purified by HPLC, as described by Chiang et al.,J. Biol. Chem. 1991, 266, 18162-18171. Results obtained withHPLC-purified material were similar to those obtained with non-HPLCpurified material.

Example 7 Oligonucleotide Synthesis—96 Well Plate Format

[0242] Oligonucleotides were synthesized via solid phase P(III)phosphoramidite chemistry on an automated synthesizer capable ofassembling 96 sequences simultaneously in a 96-well format.Phosphodiester internucleotide linkages were afforded by oxidation withaqueous iodine. Phosphorothioate internucleotide linkages were generatedby sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide(Beaucage Reagent) in anhydrous acetonitrile. Standard base-protectedbeta-cyanoethyl-diiso-propyl phosphoramidites were purchased fromcommercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., orPharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesizedas per standard or patented methods. They are utilized as base protectedbeta-cyanoethyldiisopropyl phosphoramidites.

[0243] Oligonucleotides were cleaved from support and deprotected withconcentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hoursand the released product then dried in vacuo. The dried product was thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

Example 8 Oligonucleotide Analysis—96-Well Plate Format

[0244] The concentration of oligonucleotide in each well was assessed bydilution of samples and UV absorption spectroscopy. The full-lengthintegrity of the individual products was evaluated by capillaryelectrophoresis (CE) in either the 96-well format (Beckman P/ACE™ MDQ)or, for individually prepared samples, on a commercial CE apparatus(e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition wasconfirmed by mass analysis of the compounds utilizing electrospray-massspectroscopy. All assay test plates were diluted from the master plateusing single and multi-channel robotic pipettors. Plates were judged tobe acceptable if at least 85% of the compounds on the plate were atleast 85% full length.

Example 9 Cell Culture and Oligonucleotide Treatment

[0245] The effect of antisense compounds on target nucleic acidexpression can be tested in any of a variety of cell types provided thatthe target nucleic acid is present at measurable levels. This can beroutinely determined using, for example, PCR or Northern blot analysis.The following cell types are provided for illustrative purposes, butother cell types can be routinely used, provided that the target isexpressed in the cell type chosen. This can be readily determined bymethods routine in the art, for example Northern blot analysis,ribonuclease protection assays, or RT-PCR.

[0246] T-24 Cells:

[0247] The human transitional cell bladder carcinoma cell line T-24 wasobtained from the American Type Culture Collection (ATCC) (Manassas,Va.). T-24 cells were routinely cultured in complete McCoy's 5A basalmedia (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10%fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin100 units per mL, and streptomycin 100 micrograms per mL (InvitrogenCorporation, Carlsbad, Calif.). Cells were routinely passaged bytrypsinization and dilution when they reached 90% confluence. Cells wereseeded into 96-well plates (Falcon-Primaria #3872) at a density of 7000cells/well for use in RT-PCR analysis.

[0248] For Northern blotting or other analysis, cells may be seeded onto100 mm or other standard tissue culture plates and treated similarly,using appropriate volumes of medium and oligonucleotide.

[0249] A549 Cells:

[0250] The human lung carcinoma cell line A549 was obtained from theAmerican Type Culture Collection (ATCC) (Manassas, Va.). A549 cells wereroutinely cultured in DMEM basal media (Invitrogen Corporation,Carlsbad, Calif.) supplemented with 10% fetal calf serum (InvitrogenCorporation, Carlsbad, Calif.), penicillin 100 units per mL, andstreptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad,Calif.). Cells were routinely passaged by trypsinization and dilutionwhen they reached 90% confluence.

[0251] NHDF Cells:

[0252] Human neonatal dermal fibroblast (NHDF) were obtained from theClonetics Corporation (Walkersville, Md.). NHDFs were routinelymaintained in Fibroblast Growth Medium (Clonetics Corporation,Walkersville, Md.) supplemented as recommended by the supplier. Cellswere maintained for up to 10 passages as recommended by the supplier.

[0253] HEK Cells:

[0254] Human embryonic keratinocytes (HEK) were obtained from theClonetics Corporation (Walkersville, Md.). HEKs were routinelymaintained in Keratinocyte Growth Medium (Clonetics Corporation,Walkersville, Md.) formulated as recommended by the supplier. Cells wereroutinely maintained for up to 10 passages as recommended by thesupplier.

[0255] HEPA 1-6 Cells:

[0256] The mouse hepatoma cell line HEPA 1-6 is a derivative of theBW7756 mouse hepatoma that arose in a C57/L mouse and is supplied by theAmerican Type Culture Collection (Manassas, Va.). The cells arepropagated in Dulbecco's minimal essential medium with 10% fetal bovineserum. Cells are subcultured by removing the medium, adding fresh 0.25%trypsin, 0.03% EDTA solution and letting the culture sit at roomtemperature for 3 minutes. Trypsin is then removed and the cultureallowed to at which point, fresh medium is added.

[0257] Treatment with Antisense Compounds:

[0258] When cells reached 70% confluency, they were treated witholigonucleotide. For cells grown in 96-well plates, wells were washedonce with 100 μL OPTI-MEM™-1 reduced-serum medium (InvitrogenCorporation, Carlsbad, Calif.) and then treated with 130 μL ofOPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™ (Invitrogen Corporation,Carlsbad, Calif.) and the desired concentration of oligonucleotide.After 4-7 hours of treatment, the medium was replaced with fresh medium.Cells were harvested 16-24 hours after oligonucleotide treatment.

[0259] The concentration of oligonucleotide used varies from cell lineto cell line. To determine the optimal oligonucleotide concentration fora particular cell line, the cells are treated with a positive controloligonucleotide at a range of concentrations. For human cells thepositive control oligonucleotide is selected from either ISIS 13920(TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1) which is targeted to human H-ras,or ISIS 18078, (GTGCGCGCGAGCCCGAAATC, SEQ ID NO: 2) which is targeted tohuman Jun-N-terminal kinase-2 (JNK2). Both controls are2′-O-methoxyethyl gapmers (2′-O-methoxyethyls shown in bold) with aphosphorothioate backbone. For mouse or rat cells the positive controloligonucleotide is ISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 3, a2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown in bold) with aphosphorothioate backbone which is targeted to both mouse and rat c-raf.The concentration of positive control oligonucleotide that results in80% inhibition of c-Ha-ras (for ISIS 13920), JNK2 (for ISIS 18078) orc-raf (for ISIS 15770) mRNA is then utilized as the screeningconcentration for new oligonucleotides in subsequent experiments forthat cell line. If 80% inhibition is not achieved, the lowestconcentration of positive control oligonucleotide that results in 60%inhibition of H-ras, JNK2 or c-raf mRNA is then utilized as theoligonucleotide screening concentration in subsequent experiments forthat cell line. If 60% inhibition is not achieved, that particular cellline is deemed as unsuitable for oligonucleotide transfectionexperiments. The concentrations of antisense oligonucleotides usedherein are from 50 nM to 300 nM.

Example 10 Analysis of Oligonucleotide Inhibition of LAR Expression

[0260] Antisense modulation of LAR expression can be assayed in avariety of ways known in the art. For example, LAR mRNA levels can bequantitated by, e.g., Northern blot analysis, competitive polymerasechain reaction (PCR), or real-time PCR (RT-PCR). Real-time quantitativePCR is presently preferred. RNA analysis can be performed on totalcellular RNA or poly(A)+ mRNA. The preferred method of RNA analysis ofthe present invention is the use of total cellular RNA as described inother examples herein. Methods of RNA isolation are taught in, forexample, Ausubel, F. M. et al., Current Protocols in Molecular Biology,Volume 1, pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley & Sons, Inc.,1993. Northern blot analysis is routine in the art and is taught in, forexample, Ausubel, F. M. et al., Current Protocols in Molecular Biology,Volume 1, pp. 4.2.1-4.2.9, John Wiley & Sons, Inc., 1996. Real-timequantitative (PCR) can be conveniently accomplished using thecommercially available ABI PRISM™ 7700 Sequence Detection System,available from PE-Applied Biosystems, Foster City, Calif. and usedaccording to manufacturer's instructions.

[0261] Protein levels of LAR can be quantitated in a variety of wayswell known in the art, such as immunoprecipitation, Western blotanalysis (immunoblotting), ELISA or fluorescence-activated cell sorting(FACS). Antibodies directed to LAR can be identified and obtained from avariety of sources, such as the MSRS catalog of antibodies (AerieCorporation, Birmingham, Mich.), or can be prepared via conventionalantibody generation methods. Methods for preparation of polyclonalantisera are taught in, for example, Ausubel, F. M. et al., (CurrentProtocols in Molecular Biology, Volume 2, pp. 11.12.1-11.12.9, JohnWiley & Sons, Inc., 1997). Preparation of monoclonal antibodies istaught in, for example, Ausubel, F. M. et al., (Current Protocols inMolecular Biology, Volume 2, pp. 11.4.1-11.11.5, John Wiley & Sons,Inc., 1997).

[0262] Immunoprecipitation methods are standard in the art and can befound at, for example, Ausubel, F. M. et al., (Current Protocols inMolecular Biology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons,Inc., 1998). Western blot (immunoblot) analysis is standard in the artand can be found at, for example, Ausubel, F. M. et al., (CurrentProtocols in Molecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley& Sons, Inc., 1997). Enzyme-linked immunosorbent assays (ELISA) arestandard in the art and can be found at, for example, Ausubel, F. M. etal., (Current Protocols in Molecular Biology, Volume 2, pp.11.2.1-11.2.22, John Wiley & Sons, Inc., 1991).

Example 11 Poly(A)+ mRNA Isolation

[0263] Poly(A)+ mRNA was isolated according to Miura et al., (Clin.Chem., 1996, 42, 1758-1764). Other methods for poly(A)+ mRNA isolationare taught in, for example, Ausubel, F. M. et al., (Current Protocols inMolecular Biology, Volume 1, pp. 4.5.1-4.5.3, John Wiley & Sons, Inc.,1993). Briefly, for cells grown on 96-well plates, growth medium wasremoved from the cells and each well was washed with 200 μL cold PBS. 60μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5%NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each well, theplate was gently agitated and then incubated at room temperature forfive minutes. 55 μL of lysate was transferred to Oligo d(T) coated96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated for 60minutes at room temperature, washed 3 times with 200 μL of wash buffer(10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash,the plate was blotted on paper towels to remove excess wash buffer andthen air-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH7.6), preheated to 70° C., was added to each well, the plate wasincubated on a 90° C. hot plate for 5 minutes, and the eluate was thentransferred to a fresh 96-well plate.

[0264] Cells grown on 100 mm or other standard plates may be treatedsimilarly, using appropriate volumes of all solutions.

Example 12 Total RNA Isolation

[0265] Total RNA was isolated using an RNEASY 96™ kit and bufferspurchased from Qiagen Inc. (Valencia, Calif.) following themanufacturer's recommended procedures. Briefly, for cells grown on96-well plates, growth medium was removed from the cells and each wellwas washed with 200 μL cold PBS. 150 μL Buffer RLT was added to eachwell and the plate vigorously agitated for 20 seconds. 150 μL of 70%ethanol was then added to each well and the contents mixed by pipettingthree times up and down. The samples were then transferred to the RNEASY96™ well plate attached to a QIAVAC™ manifold fitted with a wastecollection tray and attached to a vacuum source. Vacuum was applied for1 minute. 500 μL of Buffer RW1 was added to each well of the RNEASY 96™plate and incubated for 15 minutes and the vacuum was again applied for1 minute. An additional 500 μL of Buffer RW1 was added to each well ofthe RNEASY 96™ plate and the vacuum was applied for 2 minutes. 1 mL ofBuffer RPE was then added to each well of the RNEASY 96™ plate and thevacuum applied for a period of 90 seconds. The Buffer RPE wash was thenrepeated and the vacuum was applied for an additional 3 minutes. Theplate was then removed from the QIAVAC™ manifold and blotted dry onpaper towels. The plate was then re-attached to the QIAVAC™ manifoldfitted with a collection tube rack containing 1.2 mL collection tubes.RNA was then eluted by pipetting 170 μL water into each well, incubating1 minute, and then applying the vacuum for 3 minutes.

[0266] The repetitive pipetting and elution steps may be automated usinga QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially,after lysing of the cells on the culture plate, the plate is transferredto the robot deck where the pipetting, DNase treatment and elution stepsare carried out.

Example 13 Real-Time Quantitative PCR Analysis of LAR mRNA Levels

[0267] Quantitation of LAR mRNA levels was determined by real-timequantitative PCR using the ABI PRISM™ 7700 Sequence Detection System(PE-Applied Biosystems, Foster City, Calif.) according to manufacturer'sinstructions. This is a closed-tube, non-gel-based, fluorescencedetection system which allows high-throughput quantitation of polymerasechain reaction (PCR) products in real-time. As opposed to standard PCRin which amplification products are quantitated after the PCR iscompleted, products in real-time quantitative PCR are quantitated asthey accumulate. This is accomplished by including in the PCR reactionan oligonucleotide probe that anneals specifically between the forwardand reverse PCR primers, and contains two fluorescent dyes. A reporterdye (e.g., FAM or JOE, obtained from either PE-Applied Biosystems,Foster City, Calif., Operon Technologies Inc., Alameda, Calif. orIntegrated DNA Technologies Inc., Coralville, Iowa) is attached to the5′ end of the probe and a quencher dye (e.g., TAMRA, obtained fromeither PE-Applied Biosystems, Foster City, Calif., Operon TechnologiesInc., Alameda, Calif. or Integrated DNA Technologies Inc., Coralville,Iowa) is attached to the 3′ end of the probe. When the probe and dyesare intact, reporter dye emission is quenched by the proximity of the 3′quencher dye. During amplification, annealing of the probe to the targetsequence creates a substrate that can be cleaved by the 5′-exonucleaseactivity of Taq polymerase. During the extension phase of the PCRamplification cycle, cleavage of the probe by Taq polymerase releasesthe reporter dye from the remainder of the probe (and hence from thequencher moiety) and a sequence-specific fluorescent signal isgenerated. With each cycle, additional reporter dye molecules arecleaved from their respective probes, and the fluorescence intensity ismonitored at regular intervals by laser optics built into the ABI PRISM™7700 Sequence Detection System. In each assay, a series of parallelreactions containing serial dilutions of mRNA from untreated controlsamples generates a standard curve that is used to quantitate thepercent inhibition after antisense oligonucleotide treatment of testsamples.

[0268] Prior to quantitative PCR analysis, primer-probe sets specific tothe target gene being measured are evaluated for their ability to be“multiplexed” with a GAPDH amplification reaction. In multiplexing, boththe target gene and the internal standard gene GAPDH are amplifiedconcurrently in a single sample. In this analysis, mRNA isolated fromuntreated cells is serially diluted. Each dilution is amplified in thepresence of primer-probe sets specific for GAPDH only, target gene only(“single-plexing”), or both (multiplexing). Following PCR amplification,standard curves of GAPDH and target mRNA signal as a function ofdilution are generated from both the single-plexed and multiplexedsamples. If both the slope and correlation coefficient of the GAPDH andtarget signals generated from the multiplexed samples fall within 10% oftheir corresponding values generated from the single-plexed samples, theprimer-probe set specific for that target is deemed multiplexable. Othermethods of PCR are also known in the art.

[0269] PCR reagents were obtained from Invitrogen Corporation,(Carlsbad, Calif.). RT-PCR reactions were carried out by adding 20 μLPCR cocktail (2.5× PCR buffer (—MgCl2), 6.6 mM MgCl2, 375 μM each ofdATP, dCTP, dCTP and dGTP, 375 nM each of forward primer and reverseprimer, 125 nM of probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM®Taq, 5 Units MuLV reverse transcriptase, and 2.5× ROX dye) to 96-wellplates containing 30 μL total RNA solution. The RT reaction was carriedout by incubation for 30 minutes at 48° C. Following a 10 minuteincubation at 95° C. to activate the PLATINUM® Taq, 40 cycles of atwo-step PCR protocol were carried out: 95° C. for 15 seconds(denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).

[0270] Gene target quantities obtained by real time RT-PCR arenormalized using either the expression level of GAPDH, a gene whoseexpression is constant, or by quantifying total RNA using RiboGreenTM(Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression is quantifiedby real time RT-PCR, by being run simultaneously with the target,multiplexing, or separately. Total RNA is quantified using RiboGreenTMRNA quantification reagent from Molecular Probes. Methods of RNAquantification by RiboGreenTM are taught in Jones, L. J., et al,(Analytical Biochemistry, 1998, 265, 368-374).

[0271] In this assay, 170 μL of RiboGreenTM working reagent (RiboGreenTMreagent diluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipettedinto a 96-well plate containing 30 μL purified, cellular RNA. The plateis read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at480 nm and emission at 520 nm.

[0272] Probes and primers to human LAR were designed to hybridize to ahuman LAR sequence, using published sequence information (GenBankaccession number NM_(—)002840.1, incorporated herein as SEQ ID NO: 4).For human LAR the PCR primers were:

[0273] forward primer: CATCGCCATCCTCTTGTTCA (SEQ ID NO: 5)

[0274] reverse primer: CCGATCGACTGCTCATCCTT (SEQ ID NO: 6) and the

[0275] PCR probe was: FAM-AAGGAAAAGGACCCACTCTCCGTCCTC-TAMRA (SEQ ID NO:7) where FAM is the fluorescent dye and TAMRA is the quencher dye. Forhuman GAPDH the PCR primers were:

[0276] forward primer: GAAGGTGAAGGTCGGAGTC(SEQ ID NO: 8)

[0277] reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO: 9) and the

[0278] PCR probe was: 5′ JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3′ (NO: 10)where JOE is the fluorescent reporter dye and TAMRA is the quencher dye.

[0279] Probes and primers to mouse LAR were designed to hybridize to amouse LAR sequence, using published sequence information (a consensussequence constructed from Gen Bank accession numbers AW823294, AA510577,BE375498, Z37988, the complement of BE456924, AI529033, AW495458, andBE284685, AW909842 and AI385919, incorporated herein as SEQ ID NO: 11).For mouse LAR the PCR primers were:

[0280] forward primer: CTCCTGCACGGATGCTGTT (SEQ ID NO: 12)

[0281] reverse primer: GTTCCCCGAAATGCTGTGAT (SEQ ID NO: 13) and the

[0282] PCR probe was: FAM-CGGCAGAGCACAGCCCACTGG-TAMRA (SEQ ID NO: 14)where FAM is the fluorescent reporter dye and TAMRA is the quencher dye.For mouse GAPDH the PCR primers were:

[0283] forward primer: GGCAAATTCAACGGCACAGT(SEQ ID NO: 15)

[0284] reverse primer: GGGTCTCGCTCCTGGAAGAT(SEQ ID NO: 16) and the

[0285] PCR probe was: 5′ JOE-AAGGCCGAGAATGGGAAGCTTGTCATC-TAMRA 3′ (SEQID NO: 17) where JOE is the fluorescent reporter dye and TAMRA is thequencher dye.

Example 14 Northern Blot Analysis of LAR mRNA Levels

[0286] Eighteen hours after antisense treatment, cell monolayers werewashed twice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc.,Friendswood, Tex.). Total RNA was prepared following manufacturer'srecommended protocols. Twenty micrograms of total RNA was fractionatedby electrophoresis through 1.2% agarose gels containing 1.1%formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNAwas transferred from the gel to HYBOND™-N+ nylon membranes (AmershamPharmacia Biotech, Piscataway, N.J.) by overnight capillary transferusing a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc.,Friendswood, Tex.). RNA transfer was confirmed by UV visualization.Membranes were fixed by UV cross-linking using a STRATALINKER™ UVCrosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then probedusing QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.)using manufacturer's recommendations for stringent conditions.

[0287] To detect human LAR, a human LAR specific probe was prepared byPCR using the forward primer CATCGCCATCCTCTTGTTCA (SEQ ID NO: 5) and thereverse primer CCGATCGACTGCTCATCCTT (SEQ ID NO: 6). To normalize forvariations in loading and transfer efficiency membranes were strippedand probed for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH)RNA (Clontech, Palo Alto, Calif.).

[0288] To detect mouse LAR, a mouse LAR specific probe was prepared byPCR using the forward primer CTCCTGCACGGATGCTGTT (SEQ ID NO: 12) and thereverse primer GTTCCCCGAAATGCTGTGAT (SEQ ID NO: 13). To normalize forvariations in loading and transfer efficiency membranes were strippedand probed for mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH)RNA (Clontech, Palo Alto, Calif.).

[0289] Hybridized membranes were visualized and quantitated using aPHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics,Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreatedcontrols.

Example 15 Antisense Inhibition of Human LAR Expression by ChimericPhosphorothioate Oligonucleotides having 2′-MOE Wings and a Deoxy Gap

[0290] In accordance with the present invention, a series ofoligonucleotides were designed to target different regions of the humanLAR RNA, using published sequences (GenBank accession numberNM_(—)002840.1, incorporated herein as SEQ ID NO: 4, a sequenceconstructed from GenBank accession number NM_(—)002840 and thecomplement of GenBank accession number AI246688.1, incorporated hereinas SEQ ID NO: 18, GenBank accession number BE620748.1, the complement ofwhich is incorporated herein as SEQ ID NO: 19, and residues2606000-2700000 from GenBank accession number NT_(—)004852.4,incorporated herein as SEQ ID NO: 20). The oligonucleotides are shown inTable 1. “Target site” indicates the first (5′-most) nucleotide numberon the particular target sequence to which the oligonucleotide binds.All compounds in Table 1 are chimeric oligonucleotides (“gapmers”) 20nucleotides in length, composed of a central “gap” region consisting often 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′directions) by five-nucleotide “wings”. The wings are composed of2′-methoxyethyl (2′-MOE) nucleotides. The internucleoside (backbone)linkages are phosphorothioate (P═S) throughout the oligonucleotide. Allcytidine residues are 5-methylcytidines. The compounds were analyzed fortheir effect on human LAR mRNA levels by quantitative real-time PCR asdescribed in other examples herein. Data are averages from twoexperiments in which T-24 cells were treated with the oligonucleotidesof the present invention. The positive control for each datapoint isidentified in the table by sequence ID number. If present, “N.D.”indicates “no data”. TABLE 1 Inhibition of human LAR mRNA levels bychimeric phosphorothioate oligonucleotides having 2′-MOE wings and adeoxy gap TARGET CONTROL SEQ ID TARGET SEQ ID SEQ ID ISIS # REGION NOSITE SEQUENCE % INHIB NO NO 147319 Coding 4 540 gactttcttccccttcttca 5421 2 147322 Coding 4 764 ttctccaccaccttcagctg 44 22 2 147323 Coding 41027 tggagaaacgaggagccacg 61 23 2 147324 Coding 4 2194cccggaccgtggtggagccc 86 24 2 147327 Coding 4 2253 cacggagtactgggtgataa87 25 2 147328 Coding 4 2309 ctgatgccatccaccacatg 18 26 2 147330 Coding4 2385 tgtgtgtgcccgcacccaca 86 27 2 147331 Coding 4 2423accagcaccgggctgctctc 43 28 2 147333 Coding 4 2498 tgcacagcagtggagttcag84 29 2 147335 Coding 4 3346 gcatggtccgggactggatg 80 30 2 147337 Coding4 3452 aagggcacagctgacttata 51 31 2 147338 Coding 4 3509agcttccgcatcgagtgccc 57 32 2 147339 Coding 4 3605 gtgcggatggacaccaggtg83 33 2 147340 Coding 4 3725 acaatgtagaaccacctgac 54 34 2 147343 Coding4 4029 gcgcttctggtccatgggtt 79 35 2 147344 Coding 4 4167gaggatggcgatgacaatga 60 36 2 147345 Coding 4 4309 ggtggtctcgcatacctggg80 37 2 147346 Coding 4 4367 ccatcgttggctttgaggcg 85 38 2 147348 Coding4 4619 tcgcccatggtctcgggcag 72 39 2 147352 Coding 4 5042accgtcttctcgtgcttcat 65 40 2 147354 Coding 4 5537 gtcagcatgacgatgatggt86 41 2 147357 Coding 4 5703 tgtccttgactgcccatccc 72 42 2 147359 Coding4 5844 agcactgcagtgcaccgtga 82 43 2 147360 Coding 4 5904atagcgcatgcgctccagga 85 44 2 147361 Coding 4 6005 gccgcacggtagcacagctg87 45 2 147364 3′UTR 4 6111 gctcagaggagctgggtccc 85 46 2 147368 3′UTR 46312 aacagagagcttgaagcggg 74 47 2 147375 3′UTR 4 6861acctttgcaaacacgatggt 87 48 2 147378 3′UTR 4 7163 gcccccgcttggccctgagg 7349 2 147379 3′UTR 4 7202 ctaccaggcccactggcctg 28 50 2 147383 3′UTR 47327 tagtcttgccacattggttt 83 51 2 147386 3′UTR 4 7434ccacttacctatctagatag 75 52 2 147388 3′UTR 4 7514 atcttgacttagcctagcta 8453 2 147389 3′UTR 4 7562 gtaaaaatgaatgtttcatc 65 54 2 147391 3′UTR 47584 ctacagcactagcatccaca 78 55 2 147393 3′UTR 4 7629gtttttcttaacaaatagaa 37 56 2 195463 Start 18 360 gggcaccatcgtcctccctg 8557 2 Codon 195464 Coding 18 631 tggcttcatctcgctgcacc 84 58 2 195465Coding 18 1171 cgttgcggccaactggcatc 72 59 2 195466 Exon: 18 1280tttggaagagctttcactgt 59 60 2 Exon Junction 195467 Coding 18 2013ggttgggtcgaaggtgacct 64 61 2 195468 Coding 18 2087 cccatatccgagcgtgcagc44 62 2 195469 Coding 18 2722 tataggcagcaacagtaacg 66 63 2 195470 Coding18 3237 gcgggtgtctgtcgtgatgt 69 64 2 195471 Coding 18 3310cagagcctttgctggtccat 76 65 2 195472 Exon: 18 3466 tgtacagaatcttaaagggc33 66 2 Exon Junction 195473 Coding 18 3955 ggttgtagaagccccggtag 72 67 2195474 Coding 18 3986 cactggtagctcaagtccgg 75 68 2 195476 Exon: 18 4188ggtccttttccttttgaaca 79 69 2 Exon Junction 195478 Coding 18 4654tggccgtgcgctgttcccac 77 70 2 195480 Coding 18 4754 gtcacctgaataaggccaca81 71 2 195482 Coding 18 5026 tcatccgctccaacatggca 72 72 2 195484 Coding18 5074 atcgcatgcaggtcacgtgg 74 73 2 195486 Coding 18 5080tctgtgatcgcatgcaggtc 74 74 2 195488 Coding 18 5830 ccgtgataggcccatcctgt74 75 2 195490 Stop 18 6053 agcggtagttacgttgcata 85 76 2 Codon 1954923′UTR 18 6556 caatgttctgtccttcagca 77 77 2 195494 3′UTR 18 7061aagccaggctctgtgggccc 72 78 2 195496 Exon: 19 328 agccacgccctcatagcgca 8279 2 Exon Junction 195498 Exon: 20 495 ggtcactcaccgcctatcca 58 80 2Intron Junction 195500 Intron 20 19735 aactcccgggtaactccctt 61 81 2195502 Intron 20 22222 agaggcccagagaggttaag 55 82 2 195504 Intron: 2022761 actcaatgacctgtcagagg 51 83 2 Exon Junction 195506 Intron 20 60763ttgatcctcccaagagcccc 27 84 2 195508 Exon: 20 67551 gcactgcctaccgtcctcat81 85 2 Intron Junction 195510 Coding 20 90640 tccaggacgatgctcagagt 6086 2 195512 Exon: 20 90673 aacatgtcgaccacgccctc 55 87 2 Intron Junction195514 Exon: 20 90735 tctgcgttacctctgtctgc 39 88 2 Intron Junction195516 Intron: 20 91437 gatactggtcctgccaggat 25 89 2 Exon Junction195518 Coding 20 92000 ctgtccttcagcagaagtca 85 90 2 195520 Intron: 2092648 tgaaaggccagccacgcccc 62 91 2 Exon Junction

[0291] As shown in Table 1, SEQ ID NOs 23, 24, 25, 27, 29, 30, 33, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 51, 52, 53, 54,55, 57, 58, 59, 61, 63, 64, 65, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 81, 85, 86, 90 and 91 demonstrated at least 60% inhibitionof human LAR expression in this assay and are therefore preferred. Thetarget sites to which these preferred sequences are complementary areherein referred to as “preferred target regions” and are thereforepreferred sites for targeting by compounds of the present invention.These preferred target regions are shown in Table 3. The sequencesrepresent the reverse complement of the preferred antisense compoundsshown in Table 1. “Target site” indicates the first (5′-most) nucleotidenumber of the corresponding target nucleic acid. Also shown in Table 3is the species in which each of the preferred target regions was found.

Example 16 Antisense Inhibition of Mouse LAR Expression by ChimericPhosphorothioate Oligonucleotides having 2′-MOE Wings and a Deoxy Gap

[0292] In accordance with the present invention, a second series ofoligonucleotides were designed to target different regions of the mouseLAR RNA, using published sequences (a consensus sequence was constructedfrom GenBank accession numbers AW823294, AA510577, BE375498, Z37988, thecomplement of BE456924, AI529033, AW495458, and BE284685, AW909842 andAI385919, incorporated herein as SEQ ID NO: 11). The oligonucleotidesare shown in Table 2. “Target site” indicates the first (5′-most)nucleotide number on the particular target sequence to which theoligonucleotide binds. All compounds in Table 2 are chimericoligonucleotides (“gapmers”) 20 nucleotides in length, composed of acentral “gap” region consisting of ten 2′-deoxynucleotides, which isflanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”.The wings are composed of 2′-methoxyethyl (2′-MOE) nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. All cytidine residues are5-methylcytidines. The compounds were analyzed for their effect on mouseLAR mRNA levels by quantitative real-time PCR as described in otherexamples herein. Data are averages from two experiments in which HEPA1-6 cells were treated with the oligonucleotides of the presentinvention. The positive control for each datapoint is identified in thetable by sequence ID number. If present, “N.D.” indicates “no data”.TABLE 2 Inhibition of mouse LAR mRNA levels by chimeric phosphorothioateoligonucleotides having 2′-MOE wings and a deoxy gap TARGET CONTROL SEQID TARGET SEQ ID SEQ ID ISIS # REGION NO SITE SEQUENCE % INHIB NO NO147320 Coding 11 85 gctgggagctgactttcttc 0 92 1 147321 Coding 11 154gctgcactcgtaatggctgg 33 93 1 147325 Coding 11 1104 acttacccggaccgtggtgg26 94 1 147326 Coding 11 1109 acccaacttacccggaccgt 62 95 1 147329 Coding11 1223 tgctcacggctgatgccatc 35 96 1 147334 Coding 11 1408agacatgcacagcagtggag 25 97 1 147336 Coding 11 1740 cttggcaaacacttgctcca43 98 1 147341 Coding 11 2125 tcccgcccacacggtcaatg 58 99 1 147342 Coding11 2313 gcggtagctcttcttgtccc 36 100 1 147347 Coding 11 2897ccaggaacaccatcaatgga 23 101 1 147349 Coding 11 2999 ctccagaaatcgcccatggt11 102 1 147350 Coding 11 3073 cacacttcacccgggatttc 23 103 1 147351Coding 11 3193 tggagccactcttatggagg 20 104 1 147353 Coding 11 3434gtcacgtggccatagatgtc 10 105 1 147355 Coding 11 3919 cccgaagcttggtcagcatg29 106 1 147356 Coding 11 3929 ctgcccatctcccgaagctt 44 107 1 147358Coding 11 4079 cggattgtccttgactgccc 52 108 1 147362 Coding 11 4382tccagggccgcacggtagca 0 109 1 147363 Stop 11 4424 agcagtagttacgttgcata 48110 1 Codon 147365 3′UTR 11 4484 ggtatggctcagaggagctg 41 111 1 1473663′UTR 11 4585 ggctctctgactggtgtggc 40 112 1 147367 3′UTR 11 4651cacttggcccggtggacgag 21 113 1 147369 3′UTR 11 4699 gagaagcatgagaacgcgga48 114 1 147370 3′UTR 11 4895 tccttccgcagaagttgtac 50 115 1 147371 3′UTR11 4921 cacaaggaaggcgagttact 34 116 1 147372 3′UTR 11 4976agtcacgctgcctcccgggc 30 117 1 147373 3′UTR 11 4986 ggacagcaggagtcacgctg12 118 1 147374 3′UTR 11 5179 ccccagacacgtctgtggtt 30 119 1 147376 3′UTR11 5436 cccgaggtggccagaaccca 40 120 1 147377 3′UTR 11 5455tcacctgtgccattcatttc 16 121 1 147380 3′UTR 11 5546 cagacatgtgctaccaggcc21 122 1 147381 3′UTR 11 5553 ctgaggacagacatgtgcta 38 123 1 147382 3′UTR11 5586 agctgcaaaccaggagagaa 36 124 1 147384 3′UTR 11 5667aagtccagtagtcttgccac 44 125 1 147385 3′UTR 11 5742 tgcccagaggaagagaccct62 126 1 147337 3′UTR 11 5790 aacagctatgcacccttccc 30 127 1 147390 3′UTR11 5899 gcatccacaaggtaaaaatg 29 128 1 147392 3′UTR 11 5920acagtgaactctacagcact 59 129 1 147394 3′UTR 11 5968 catgatcgctgtagtttttc0 130 1 147395 3′UTR 11 6044 agatacagagctgagacaga 5 131 1 147396 3′UTR11 6103 ggctcacccccttgggagga 0 132 1

[0293] As shown in Table 2, SEQ ID NOs 95, 98, 99, 107, 108, 110, 111,112, 114, 115, 120, 125, 126 and 129 demonstrated at least 40%inhibition of mouse LAR expression in this experiment and are thereforepreferred. The target sites to which these preferred sequences arecomplementary are herein referred to as “preferred target regions” andare therefore preferred sites for targeting by compounds of the presentinvention. These preferred target regions are shown in Table 3. Thesequences represent the reverse complement of the preferred antisensecompounds shown in Table 1. “Target sites” indicates the first (5′-most)nucleotide number of the corresponding target nucleic acid. Also shownin Table 3 is the species in which each of the preferred target regionswas found. TABLE 3 Sequence and position of preferred target regionsidentified in LAR. TARGET REV COMP SEQ ID TARGET OF SEQ SEQ ID SITEID NOSITE SEQUENCE ID ACTIVE IN NO 62564 4 1027 Cgtggctcctcgtttctcca 23 H.sapiens 133 62565 4 2194 Gggctccaccacggtccggg 24 H. sapiens 134 62568 42253 Ttatcacccagtactccgtg 25 H. sapiens 135 62571 4 2385tgtgggtgcgggcacacaca 27 H. sapiens 136 62574 4 2498 ctgaactccactgctgtgca29 H. sapiens 137 62576 4 3346 catccagtcccggaccatgc 30 H. sapiens 13862580 4 3605 cacctggtgtccatccgcac 33 H. sapiens 139 62584 4 4029aacccatggaccagaagcgc 35 H. sapiens 140 62585 4 4167 tcattgtcatcgccatcctc36 H. sapiens 141 62586 4 4309 cccaggtatgcgagaccacc 37 H. sapiens 14262587 4 4367 cgcctcaaagccaacgatgg 38 H. sapiens 143 62589 4 4619ctgcccgagaccatgggcga 39 H. sapiens 144 62593 4 5042 atgaagcacgagaagacggt40 H. sapiens 145 62595 4 5537 accatcatcgtcatgctgac 41 H. sapiens 14662598 4 5703 gggatgggcagtcaaggaca 42 H. sapiens 147 62600 4 5844tcacggtgcactgcagtgct 43 H. sapiens 148 62601 4 5904 tcctggagcgcatgcgctat44 H. sapiens 149 62602 4 6005 cagctgtgctaccgtgcqgc 45 H. sapiens 15062605 4 6111 gggacccagctcctctgagc 46 H. sapiens 151 62609 4 6312cccgcttcaagctctctgtt 47 H. sapiens 152 62616 4 6861 accatcgtgtttgcaaaggt48 H. sapiens 153 62619 4 7163 cctcagggccaagcgggqgc 49 H. sapiens 15462624 4 7327 aaaccaatgtggcaagacta 51 H. sapiens 155 62627 4 7434ctatctagataggtaagtgg 52 H. sapiens 156 62629 4 7514 tagctaggctaagtcaagat53 H. sapiens 157 62630 4 7562 gatgaaacattcatttttac 54 H. sapiens 15862632 4 7584 tgtggatgctagtgctgtag 55 H. sapiens 159 113695 18 360cagggaggacgatggtgccc 57 H. sapiens 160 113696 18 631ggtgcagcgagatgaagcca 58 H. sapiens 161 113697 18 1171gatgccagttggccgcaacg 59 H. sapiens 162 113699 18 2013aggtcaccttcgacccaacc 61 H. sapiens 163 113701 18 2722cgttactgttgctgcctata 63 H. sapiens 164 113702 18 3237acatcacgacagacacccgc 64 H. sapiens 165 113703 18 3310atggaccagcaaaggctctg 65 H. sapiens 166 113705 18 3955ctaccggggcttctacaacc 67 H. sapiens 167 113706 18 3986ccggacttgagctaccagtg 68 H. sapiens 168 113707 18 4188tgttcaaaaggaaaaggacc 69 H. sapiens 169 113708 18 4654gtgggaacagcgcacggcca 70 H. sapiens 170 113709 18 4754tgtggccttattcaggtgac 71 H. sapiens 171 113710 18 5026tgccatgttggagcggatga 72 H. sapiens 172 113711 18 5074ccacgtgacctgcatgcgat 73 H. sapiens 173 113712 18 5080gacctgcatgcgatcacaga 74 H. sapiens 174 113713 18 5830acaggatgggcctatcacgg 75 H. sapiens 175 113714 18 6053tatgcaacgtaactaccgct 76 H. sapiens 176 113715 18 6556tgctgaaggacagaacattg 77 H. sapiens 177 113716 18 7061gqgcccacagagcctggctt 78 H. sapiens 178 113717 19 328tgcgctatgagggcgtggct 79 H. sapiens 179 113719 20 19735aagggaqttacccgggagtt 81 H. sapiens 180 113723 20 67551atgaggacggtaggcagtgc 85 H. sapiens 181 113724 20 90640actctgagcatcgtcctgga 86 H. sapiens 182 113728 20 92000tgacttctgctgaaggacag 90 H. sapiens 183 113729 20 92648ggggcgtggctggcctttca 91 H. sapiens 184 62567 11 1109acggtccgggtaagttgggt 95 M. musculus 185 62577 11 1740tggagcaagtgtttgccaag 98 M. musculus 186 62582 11 2125cattgaccgtgtgggcggga 99 M. musculus 187 62597 11 3929aagcttcgggagatgggcag 107 M. musculus 188 62599 11 4079gggcagtcaaggacaatccg 108 M. musculus 189 62604 11 4424tatqcaacgtaactactgct 110 M. musculus 190 62606 11 4484cagctcctctgagccatacc 111 M. musculus 191 62607 11 4585gccacaccagtcagagagcc 112 M. musculus 192 62610 11 4699tccgcgttctcatgcttctc 114 M. musculus 193 62611 11 4895gtacaacttctgcggaagga 115 M. musculus 194 62617 11 5436tgggttctggccacctcggg 120 H. musculus 195 62625 11 5667gtggcaagactactggactt 125 M. musculus 196 62626 11 5742agggtctcttcctctgggca 126 M. musculus 197 62633 11 5920agtgctgtagagttcaccgt 129 M. musculus 198

[0294] As these “preferred target regions” have been found byexperimentation to be open to, and accessible for, hybridization withthe antisense compounds of the present invention, one of skill in theart will recognize or be able to ascertain, using no more than routineexperimentation, further embodiments of the invention that encompassother compounds that specifically hybridize to these sites andconsequently inhibit the expression of LAR.

[0295] In one embodiment, the “preferred target region” may be employedin screening candidate antisense compounds. “Candidate antisensecompounds” are those that inhibit the expression of a nucleic acidmolecule encoding LAR and which comprise at least an 8-nucleobaseportion which is complementary to a preferred target region. The methodcomprises the steps of contacting a preferred target region of a nucleicacid molecule encoding LAR with one or more candidate antisensecompounds, and selecting for one or more candidate antisense compoundswhich inhibit the expression of a nucleic acid molecule encoding LAR.Once it is shown that the candidate antisense compound or compounds arecapable of inhibiting the expression of a nucleic acid molecule encodingLAR, the candidate antisense compound may be employed as an antisensecompound in accordance with the present invention.

[0296] According to the present invention, antisense compounds includeribozymes, external guide sequence (EGS) oligonucleotides (oligozymes),and other short catalytic RNAs or catalytic oligonucleotides whichhybridize to the target nucleic acid and modulate its expression.

Example 17 Western Blot Analysis of LAR Protein Levels

[0297] Western blot analysis (immunoblot analysis) is carried out usingstandard methods. Cells are harvested 16-20 h after oligonucleotidetreatment, washed once with PBS, suspended in Laemmli buffer (100ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gelsare run for 1.5 hours at 150 V, and transferred to membrane for westernblotting. Appropriate primary antibody directed to LAR is used, with aradiolabeled or fluorescently labeled secondary antibody directedagainst the primary antibody species. Bands are visualized using aPHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.).

1 198 1 20 DNA Artificial Sequence Antisense Oligonucleotide 1tccgtcatcg ctcctcaggg 20 2 20 DNA Artificial Sequence AntisenseOligonucleotide 2 gtgcgcgcga gcccgaaatc 20 3 20 DNA Artificial SequenceAntisense Oligonucleotide 3 atgcattctg cccccaagga 20 4 7702 DNA H.sapiens CDS (371)...(6064) 4 cgggagcggc gggagcggtg gcggcggcag aggcggcggctccagcttcg gctccggctc 60 gggctcgggc tccggctccg gctccggctc cggctccagctcgggtggcg gtggcgggag 120 cgggaccagg tggaggcggc ggcggcagag gagtgggagcagcggcccta gcggcttgcg 180 gggggacatg cggaccgacg gcccctggat aggcggaaggagtggaggcc ctggtgcccg 240 gcccttggtg ctgagtatcc agcaagagtg accggggtgaagaagcaaag actcggttga 300 ttgtcctggg ctgtggctgg ctgtggagct agagccctggatggcccctg agccagcccc 360 agggaggacg atg gtg ccc ctt gtg cct gca ctg gtgatg ctt ggt ttg 409 Met Val Pro Leu Val Pro Ala Leu Val Met Leu Gly Leu1 5 10 gtg gca ggc gcc cat ggt gac agc aaa cct gtc ttc att aaa gtc cct457 Val Ala Gly Ala His Gly Asp Ser Lys Pro Val Phe Ile Lys Val Pro 1520 25 gag gac cag act ggg ctg tca gga ggg gta gcc tcc ttc gtg tgc caa505 Glu Asp Gln Thr Gly Leu Ser Gly Gly Val Ala Ser Phe Val Cys Gln 3035 40 45 gct aca gga gaa ccc aag ccg cgc atc aca tgg atg aag aag ggg aag553 Ala Thr Gly Glu Pro Lys Pro Arg Ile Thr Trp Met Lys Lys Gly Lys 5055 60 aaa gtc agc tcc cag cgc ttc gag gtc att gag ttt gat gat ggg gca601 Lys Val Ser Ser Gln Arg Phe Glu Val Ile Glu Phe Asp Asp Gly Ala 6570 75 ggg tca gtg ctt cgg atc cag cca ttg cgg gtg cag cga gat gaa gcc649 Gly Ser Val Leu Arg Ile Gln Pro Leu Arg Val Gln Arg Asp Glu Ala 8085 90 atc tat gag tgt aca gct act aac agc ctg ggt gag atc aac act agt697 Ile Tyr Glu Cys Thr Ala Thr Asn Ser Leu Gly Glu Ile Asn Thr Ser 95100 105 gcc aag ctc tca gtg ctc gaa gag gaa cag ctg ccc cct ggg ttc cct745 Ala Lys Leu Ser Val Leu Glu Glu Glu Gln Leu Pro Pro Gly Phe Pro 110115 120 125 tcc atc gac atg ggg cct cag ctg aag gtg gtg gag aag gca cgcaca 793 Ser Ile Asp Met Gly Pro Gln Leu Lys Val Val Glu Lys Ala Arg Thr130 135 140 gcc acc atg cta tgt gcc gca ggc gga aat cca gac cct gag atttct 841 Ala Thr Met Leu Cys Ala Ala Gly Gly Asn Pro Asp Pro Glu Ile Ser145 150 155 tgg ttc aag gac ttc ctt cct gta gac cct gcc acg agc aac ggccgc 889 Trp Phe Lys Asp Phe Leu Pro Val Asp Pro Ala Thr Ser Asn Gly Arg160 165 170 atc aag cag ctg cgt tca ggt gcc ttg cag ata gag agc agt gaggaa 937 Ile Lys Gln Leu Arg Ser Gly Ala Leu Gln Ile Glu Ser Ser Glu Glu175 180 185 tcc gac caa ggc aag tac gag tgt gtg gcg acc aac tcg gca ggcaca 985 Ser Asp Gln Gly Lys Tyr Glu Cys Val Ala Thr Asn Ser Ala Gly Thr190 195 200 205 cgt tac tca gcc cct gcg aac ctg tat gtg cga gtg cgc cgcgtg gct 1033 Arg Tyr Ser Ala Pro Ala Asn Leu Tyr Val Arg Val Arg Arg ValAla 210 215 220 cct cgt ttc tcc atc cct ccc agc agc cag gag gtg atg ccaggc ggc 1081 Pro Arg Phe Ser Ile Pro Pro Ser Ser Gln Glu Val Met Pro GlyGly 225 230 235 agc gtg aac ctg aca tgc gtg gca gtg ggt gca ccc atg ccctac gtg 1129 Ser Val Asn Leu Thr Cys Val Ala Val Gly Ala Pro Met Pro TyrVal 240 245 250 aag tgg atg atg ggg gcc gag gag ctc acc aag gag gat gagatg cca 1177 Lys Trp Met Met Gly Ala Glu Glu Leu Thr Lys Glu Asp Glu MetPro 255 260 265 gtt ggc cgc aac gtc ctg gag ctc agc aat gtc gta cgc tctgcc aac 1225 Val Gly Arg Asn Val Leu Glu Leu Ser Asn Val Val Arg Ser AlaAsn 270 275 280 285 tac acc tgt gtg gcc atc tcc tcg ctg ggc atg atc gaggcc aca gcc 1273 Tyr Thr Cys Val Ala Ile Ser Ser Leu Gly Met Ile Glu AlaThr Ala 290 295 300 cag gtc aca gtg aaa gct ctt cca aag cct ccg att gatctt gtg gtg 1321 Gln Val Thr Val Lys Ala Leu Pro Lys Pro Pro Ile Asp LeuVal Val 305 310 315 aca gag aca act gcc acc agt gtc acc ctc acc tgg gactct ggg aac 1369 Thr Glu Thr Thr Ala Thr Ser Val Thr Leu Thr Trp Asp SerGly Asn 320 325 330 tcg gag cct gta acc tac tat ggc atc cag tac cgc gcagcg ggc acg 1417 Ser Glu Pro Val Thr Tyr Tyr Gly Ile Gln Tyr Arg Ala AlaGly Thr 335 340 345 gag ggc ccc ttt cag gag gtg gat ggt gtg gcc acc acccgc tac agc 1465 Glu Gly Pro Phe Gln Glu Val Asp Gly Val Ala Thr Thr ArgTyr Ser 350 355 360 365 att ggc ggc ctc agc cct ttc tcg gaa tat gcc ttccgc gtg ctg gcg 1513 Ile Gly Gly Leu Ser Pro Phe Ser Glu Tyr Ala Phe ArgVal Leu Ala 370 375 380 gtg aac agc atc ggg cga ggg ccg ccc agc gag gcagtg cgg gca cgc 1561 Val Asn Ser Ile Gly Arg Gly Pro Pro Ser Glu Ala ValArg Ala Arg 385 390 395 acg gga gaa cag gcg ccc tcc agc cca ccg cgc cgcgtg cag gca cgc 1609 Thr Gly Glu Gln Ala Pro Ser Ser Pro Pro Arg Arg ValGln Ala Arg 400 405 410 atg ctg agc gcc agc acc atg ctg gtg cag tgg gagcct ccc gag gag 1657 Met Leu Ser Ala Ser Thr Met Leu Val Gln Trp Glu ProPro Glu Glu 415 420 425 ccc aac ggc ctg gtg cgg gga tac cgc gtc tac tatact ccg gac tcc 1705 Pro Asn Gly Leu Val Arg Gly Tyr Arg Val Tyr Tyr ThrPro Asp Ser 430 435 440 445 cgc cgc ccc ccg aac gcc tgg cac aag cac aacacc gac gcg ggg ctc 1753 Arg Arg Pro Pro Asn Ala Trp His Lys His Asn ThrAsp Ala Gly Leu 450 455 460 ctc acg acc gtg ggc agc ctg ctg cct ggc atcacc tac agc ctg cgc 1801 Leu Thr Thr Val Gly Ser Leu Leu Pro Gly Ile ThrTyr Ser Leu Arg 465 470 475 gtg ctt gcc ttc acc gcc gtg ggc gat ggc cctccc agc ccc acc atc 1849 Val Leu Ala Phe Thr Ala Val Gly Asp Gly Pro ProSer Pro Thr Ile 480 485 490 cag gtc aag acg cag cag gga gtg cct gcc cagccc gcg gac ttc cag 1897 Gln Val Lys Thr Gln Gln Gly Val Pro Ala Gln ProAla Asp Phe Gln 495 500 505 gcc gag gtg gag tcg gac acc agg atc cag ctctcg tgg ctg ctg ccc 1945 Ala Glu Val Glu Ser Asp Thr Arg Ile Gln Leu SerTrp Leu Leu Pro 510 515 520 525 cct cag gag cgg atc atc atg tat gaa ctggtg tac tgg gcg gca gag 1993 Pro Gln Glu Arg Ile Ile Met Tyr Glu Leu ValTyr Trp Ala Ala Glu 530 535 540 gac gaa gac caa cag cac aag gtc acc ttcgac cca acc tcc tcc tac 2041 Asp Glu Asp Gln Gln His Lys Val Thr Phe AspPro Thr Ser Ser Tyr 545 550 555 aca cta gag gac ctg aag cct gac aca ctctac cgc ttc cag ctg gct 2089 Thr Leu Glu Asp Leu Lys Pro Asp Thr Leu TyrArg Phe Gln Leu Ala 560 565 570 gca cgc tcg gat atg ggg gtg ggc gtc ttcacc ccc acc att gag gcc 2137 Ala Arg Ser Asp Met Gly Val Gly Val Phe ThrPro Thr Ile Glu Ala 575 580 585 cgc aca gcc cag tcc acc ccc tcc gcc cctccc cag aag gtg atg tgt 2185 Arg Thr Ala Gln Ser Thr Pro Ser Ala Pro ProGln Lys Val Met Cys 590 595 600 605 gtg agc atg ggc tcc acc acg gtc cgggta agt tgg gtc ccg ccg cct 2233 Val Ser Met Gly Ser Thr Thr Val Arg ValSer Trp Val Pro Pro Pro 610 615 620 gcc gac agc cgc aac ggc gtt atc acccag tac tcc gtg gcc cac gag 2281 Ala Asp Ser Arg Asn Gly Val Ile Thr GlnTyr Ser Val Ala His Glu 625 630 635 gcg gtg gac ggc gag gac cgc ggg cggcat gtg gtg gat ggc atc agc 2329 Ala Val Asp Gly Glu Asp Arg Gly Arg HisVal Val Asp Gly Ile Ser 640 645 650 cgt gag cac tcc agc tgg gac ctg gtgggc ctg gag aag tgg acg gag 2377 Arg Glu His Ser Ser Trp Asp Leu Val GlyLeu Glu Lys Trp Thr Glu 655 660 665 tac cgg gtg tgg gtg cgg gca cac acagac gtg ggc ccc ggc ccc gag 2425 Tyr Arg Val Trp Val Arg Ala His Thr AspVal Gly Pro Gly Pro Glu 670 675 680 685 agc agc ccg gtg ctg gtg cgc accgat gag gac gtg ccc agc ggg cct 2473 Ser Ser Pro Val Leu Val Arg Thr AspGlu Asp Val Pro Ser Gly Pro 690 695 700 ccg cgg aag gtg gag gtg gag ccactg aac tcc act gct gtg cat gtc 2521 Pro Arg Lys Val Glu Val Glu Pro LeuAsn Ser Thr Ala Val His Val 705 710 715 tac tgg aag ctg cct gtc ccc agcaag cag cat ggc cag atc cgc ggc 2569 Tyr Trp Lys Leu Pro Val Pro Ser LysGln His Gly Gln Ile Arg Gly 720 725 730 tac cag gtc acc tac gtg cgg ctggag aat ggc gag ccc cgt gga ctc 2617 Tyr Gln Val Thr Tyr Val Arg Leu GluAsn Gly Glu Pro Arg Gly Leu 735 740 745 ccc atc atc caa gac gtc atg ctagcc gag gcc cag tgg cgg cca gag 2665 Pro Ile Ile Gln Asp Val Met Leu AlaGlu Ala Gln Trp Arg Pro Glu 750 755 760 765 gag tcc gag gac tat gaa accact atc agc ggc ctg acc ccg gag acc 2713 Glu Ser Glu Asp Tyr Glu Thr ThrIle Ser Gly Leu Thr Pro Glu Thr 770 775 780 acc tac tcc gtt act gtt gctgcc tat acc acc aag ggg gat ggt gcc 2761 Thr Tyr Ser Val Thr Val Ala AlaTyr Thr Thr Lys Gly Asp Gly Ala 785 790 795 cgc agc aag ccc aaa att gtcact aca aca ggt gca gtc cca ggc cgg 2809 Arg Ser Lys Pro Lys Ile Val ThrThr Thr Gly Ala Val Pro Gly Arg 800 805 810 ccc acc atg atg atc agc accacg gcc atg aac act gcg ctg ctc cag 2857 Pro Thr Met Met Ile Ser Thr ThrAla Met Asn Thr Ala Leu Leu Gln 815 820 825 tgg cac cca ccc aag gaa ctgcct ggc gag ctg ctg ggc tac cgg ctg 2905 Trp His Pro Pro Lys Glu Leu ProGly Glu Leu Leu Gly Tyr Arg Leu 830 835 840 845 cag tac tgc cgg gcc gacgag gcg cgg ccc aac acc ata gat ttc ggc 2953 Gln Tyr Cys Arg Ala Asp GluAla Arg Pro Asn Thr Ile Asp Phe Gly 850 855 860 aag gat gac cag cac ttcaca gtc acc ggc ctg cac aag ggg acc acc 3001 Lys Asp Asp Gln His Phe ThrVal Thr Gly Leu His Lys Gly Thr Thr 865 870 875 tac atc ttc cgg ctt gctgcc aag aac cgg gct ggc ttg ggt gag gag 3049 Tyr Ile Phe Arg Leu Ala AlaLys Asn Arg Ala Gly Leu Gly Glu Glu 880 885 890 ttc gag aag gag atc aggacc ccc gag gac ctg ccc agc ggc ttc ccc 3097 Phe Glu Lys Glu Ile Arg ThrPro Glu Asp Leu Pro Ser Gly Phe Pro 895 900 905 caa aac ctg cat gtg acagga ctg acc acg tct acc aca gaa ctg gcc 3145 Gln Asn Leu His Val Thr GlyLeu Thr Thr Ser Thr Thr Glu Leu Ala 910 915 920 925 tgg gac ccg cca gtgctg gcg gag agg aac ggg cgc atc atc agc tac 3193 Trp Asp Pro Pro Val LeuAla Glu Arg Asn Gly Arg Ile Ile Ser Tyr 930 935 940 acc gtg gtg ttc cgagac atc aac agc caa cag gag ctg cag aac atc 3241 Thr Val Val Phe Arg AspIle Asn Ser Gln Gln Glu Leu Gln Asn Ile 945 950 955 acg aca gac acc cgcttt acc ctt act ggc ctc aag cca gac acc act 3289 Thr Thr Asp Thr Arg PheThr Leu Thr Gly Leu Lys Pro Asp Thr Thr 960 965 970 tac gac atc aag gtccgc gca tgg acc agc aaa ggc tct ggc cca ctc 3337 Tyr Asp Ile Lys Val ArgAla Trp Thr Ser Lys Gly Ser Gly Pro Leu 975 980 985 agc ccc agc atc cagtcc cgg acc atg ccg gtg gag caa gtg ttt gcc 3385 Ser Pro Ser Ile Gln SerArg Thr Met Pro Val Glu Gln Val Phe Ala 990 995 1000 1005 aag aac ttccgg gtg gcg gct gca atg aag acg tct gtg ctg ctc agc 3433 Lys Asn Phe ArgVal Ala Ala Ala Met Lys Thr Ser Val Leu Leu Ser 1010 1015 1020 tgg gaggtt ccc gac tcc tat aag tca gct gtg ccc ttt aag att ctg 3481 Trp Glu ValPro Asp Ser Tyr Lys Ser Ala Val Pro Phe Lys Ile Leu 1025 1030 1035 tacaat ggg cag agt gtg gag gtg gac ggg cac tcg atg cgg aag ctg 3529 Tyr AsnGly Gln Ser Val Glu Val Asp Gly His Ser Met Arg Lys Leu 1040 1045 1050atc gca gac ctg cag ccc aac aca gag tac tcg ttt gtg ctg atg aac 3577 IleAla Asp Leu Gln Pro Asn Thr Glu Tyr Ser Phe Val Leu Met Asn 1055 10601065 cgt ggc agc agc gca ggg ggc ctg cag cac ctg gtg tcc atc cgc aca3625 Arg Gly Ser Ser Ala Gly Gly Leu Gln His Leu Val Ser Ile Arg Thr1070 1075 1080 1085 gcc ccc gac ctc ctg cct cac aag ccg ctg cct gcc tctgcc tac ata 3673 Ala Pro Asp Leu Leu Pro His Lys Pro Leu Pro Ala Ser AlaTyr Ile 1090 1095 1100 gag gac ggc cgc ttc gat ctc tcc atg ccc cat gtgcaa gac ccc tcg 3721 Glu Asp Gly Arg Phe Asp Leu Ser Met Pro His Val GlnAsp Pro Ser 1105 1110 1115 ctt gtc agg tgg ttc tac att gtt gtg gta cccatt gac cgt gtg ggc 3769 Leu Val Arg Trp Phe Tyr Ile Val Val Val Pro IleAsp Arg Val Gly 1120 1125 1130 ggg agc atg ctg acg cca agg tgg agc acaccc gag gaa ctg gag ctg 3817 Gly Ser Met Leu Thr Pro Arg Trp Ser Thr ProGlu Glu Leu Glu Leu 1135 1140 1145 gac gag ctt cta gaa gcc atc gag caaggc gga gag gag cag cgg cgg 3865 Asp Glu Leu Leu Glu Ala Ile Glu Gln GlyGly Glu Glu Gln Arg Arg 1150 1155 1160 1165 cgg cgg cgg cag gca gaa cgtctg aag cca tat gtg gct gct caa ctg 3913 Arg Arg Arg Gln Ala Glu Arg LeuLys Pro Tyr Val Ala Ala Gln Leu 1170 1175 1180 gat gtg ctc ccg gag accttt acc ttg ggg gac aag aag aac tac cgg 3961 Asp Val Leu Pro Glu Thr PheThr Leu Gly Asp Lys Lys Asn Tyr Arg 1185 1190 1195 ggc ttc tac aac cggccc ctg tct ccg gac ttg agc tac cag tgc ttt 4009 Gly Phe Tyr Asn Arg ProLeu Ser Pro Asp Leu Ser Tyr Gln Cys Phe 1200 1205 1210 gtg ctt gcc tccttg aag gaa ccc atg gac cag aag cgc tat gcc tcc 4057 Val Leu Ala Ser LeuLys Glu Pro Met Asp Gln Lys Arg Tyr Ala Ser 1215 1220 1225 agc ccc tactcg gat gag atc gtg gtc cag gtg aca cca gcc cag cag 4105 Ser Pro Tyr SerAsp Glu Ile Val Val Gln Val Thr Pro Ala Gln Gln 1230 1235 1240 1245 caggag gag ccg gag atg ctg tgg gtg acg ggt ccc gtg ctg gca gtc 4153 Gln GluGlu Pro Glu Met Leu Trp Val Thr Gly Pro Val Leu Ala Val 1250 1255 1260atc ctc atc atc ctc att gtc atc gcc atc ctc ttg ttc aaa agg aaa 4201 IleLeu Ile Ile Leu Ile Val Ile Ala Ile Leu Leu Phe Lys Arg Lys 1265 12701275 agg acc cac tct ccg tcc tct aag gat gag cag tcg atc gga ctg aag4249 Arg Thr His Ser Pro Ser Ser Lys Asp Glu Gln Ser Ile Gly Leu Lys1280 1285 1290 gac tcc ttg ctg gcc cac tcc tct gac cct gtg gag atg cggagg ctc 4297 Asp Ser Leu Leu Ala His Ser Ser Asp Pro Val Glu Met Arg ArgLeu 1295 1300 1305 aac tac cag acc cca ggt atg cga gac cac cca ccc atcccc atc acc 4345 Asn Tyr Gln Thr Pro Gly Met Arg Asp His Pro Pro Ile ProIle Thr 1310 1315 1320 1325 gac ctg gcg gac aac atc gag cgc ctc aaa gccaac gat ggc ctc aag 4393 Asp Leu Ala Asp Asn Ile Glu Arg Leu Lys Ala AsnAsp Gly Leu Lys 1330 1335 1340 ttc tcc cag gag tat gag tcc atc gac cctgga cag cag ttc acg tgg 4441 Phe Ser Gln Glu Tyr Glu Ser Ile Asp Pro GlyGln Gln Phe Thr Trp 1345 1350 1355 gag aat tca aac ctg gag gtg aac aagccc aag aac cgc tat gcg aat 4489 Glu Asn Ser Asn Leu Glu Val Asn Lys ProLys Asn Arg Tyr Ala Asn 1360 1365 1370 gtc atc gcc tac gac cac tct cgagtc atc ctt acc tct atc gat ggc 4537 Val Ile Ala Tyr Asp His Ser Arg ValIle Leu Thr Ser Ile Asp Gly 1375 1380 1385 gtc ccc ggg agt gac tac atcaat gcc aac tac atc gat ggc tac cgc 4585 Val Pro Gly Ser Asp Tyr Ile AsnAla Asn Tyr Ile Asp Gly Tyr Arg 1390 1395 1400 1405 aag cag aat gcc tacatc gcc acg cag ggc ccc ctg ccc gag acc atg 4633 Lys Gln Asn Ala Tyr IleAla Thr Gln Gly Pro Leu Pro Glu Thr Met 1410 1415 1420 ggc gat ttc tggaga atg gtg tgg gaa cag cgc acg gcc act gtg gtc 4681 Gly Asp Phe Trp ArgMet Val Trp Glu Gln Arg Thr Ala Thr Val Val 1425 1430 1435 atg atg acacgg ctg gag gag aag tcc cgg gta aaa tgt gat cag tac 4729 Met Met Thr ArgLeu Glu Glu Lys Ser Arg Val Lys Cys Asp Gln Tyr 1440 1445 1450 tgg ccagcc cgt ggc acc gag acc tgt ggc ctt att cag gtg acc ctg 4777 Trp Pro AlaArg Gly Thr Glu Thr Cys Gly Leu Ile Gln Val Thr Leu 1455 1460 1465 ttggac aca gtg gag ctg gcc aca tac act gtg cgc acc ttc gca ctc 4825 Leu AspThr Val Glu Leu Ala Thr Tyr Thr Val Arg Thr Phe Ala Leu 1470 1475 14801485 cac aag agt ggc tcc agt gag aag cgt gag ctg cgt cag ttt cag ttc4873 His Lys Ser Gly Ser Ser Glu Lys Arg Glu Leu Arg Gln Phe Gln Phe1490 1495 1500 atg gcc tgg cca gac cat gga gtt cct gag tac cca act cccatc ctg 4921 Met Ala Trp Pro Asp His Gly Val Pro Glu Tyr Pro Thr Pro IleLeu 1505 1510 1515 gcc ttc cta cga cgg gtc aag gcc tgc aac ccc cta gacgca ggg ccc 4969 Ala Phe Leu Arg Arg Val Lys Ala Cys Asn Pro Leu Asp AlaGly Pro 1520 1525 1530 atg gtg gtg cac tgc agc gcg ggc gtg ggc cgc accggc tgc ttc atc 5017 Met Val Val His Cys Ser Ala Gly Val Gly Arg Thr GlyCys Phe Ile 1535 1540 1545 gtg att gat gcc atg ttg gag cgg atg aag cacgag aag acg gtg gac 5065 Val Ile Asp Ala Met Leu Glu Arg Met Lys His GluLys Thr Val Asp 1550 1555 1560 1565 atc tat ggc cac gtg acc tgc atg cgatca cag agg aac tac atg gtg 5113 Ile Tyr Gly His Val Thr Cys Met Arg SerGln Arg Asn Tyr Met Val 1570 1575 1580 cag acg gag gac cag tac gtg ttcatc cat gag gcg ctg ctg gag gct 5161 Gln Thr Glu Asp Gln Tyr Val Phe IleHis Glu Ala Leu Leu Glu Ala 1585 1590 1595 gcc acg tgc ggc cac aca gaggtg cct gcc cgc aac ctg tat gcc cac 5209 Ala Thr Cys Gly His Thr Glu ValPro Ala Arg Asn Leu Tyr Ala His 1600 1605 1610 atc cag aag ctg ggc caagtg cct cca ggg gag agt gtg acc gcc atg 5257 Ile Gln Lys Leu Gly Gln ValPro Pro Gly Glu Ser Val Thr Ala Met 1615 1620 1625 gag ctc gag ttc aagttg ctg gcc agc tcc aag gcc cac acg tcc cgc 5305 Glu Leu Glu Phe Lys LeuLeu Ala Ser Ser Lys Ala His Thr Ser Arg 1630 1635 1640 1645 ttc atc agcgcc aac ctg ccc tgc aac aag ttc aag aac cgg ctg gtg 5353 Phe Ile Ser AlaAsn Leu Pro Cys Asn Lys Phe Lys Asn Arg Leu Val 1650 1655 1660 aac atcatg ccc tac gaa ttg acc cgt gtg tgt ctg cag ccc atc cgt 5401 Asn Ile MetPro Tyr Glu Leu Thr Arg Val Cys Leu Gln Pro Ile Arg 1665 1670 1675 ggtgtg gag ggc tct gac tac atc aat gcc agc ttc ctg gat ggt tat 5449 Gly ValGlu Gly Ser Asp Tyr Ile Asn Ala Ser Phe Leu Asp Gly Tyr 1680 1685 1690aga cag cag aag gcc tac ata gct aca cag ggg cct ctg gca gag agc 5497 ArgGln Gln Lys Ala Tyr Ile Ala Thr Gln Gly Pro Leu Ala Glu Ser 1695 17001705 acc gag gac ttc tgg cgc atg cta tgg gag cac aat tcc acc atc atc5545 Thr Glu Asp Phe Trp Arg Met Leu Trp Glu His Asn Ser Thr Ile Ile1710 1715 1720 1725 gtc atg ctg acc aag ctt cgg gag atg ggc agg gag aaatgc cac cag 5593 Val Met Leu Thr Lys Leu Arg Glu Met Gly Arg Glu Lys CysHis Gln 1730 1735 1740 tac tgg cca gca gag cgc tct gct cgc tac cag tacttt gtt gtt gac 5641 Tyr Trp Pro Ala Glu Arg Ser Ala Arg Tyr Gln Tyr PheVal Val Asp 1745 1750 1755 ccg atg gct gag tac aac atg ccc cag tat atcctg cgt gag ttc aag 5689 Pro Met Ala Glu Tyr Asn Met Pro Gln Tyr Ile LeuArg Glu Phe Lys 1760 1765 1770 gtc acg gat gcc cgg gat ggg cag tca aggaca atc cgg cag ttc cag 5737 Val Thr Asp Ala Arg Asp Gly Gln Ser Arg ThrIle Arg Gln Phe Gln 1775 1780 1785 ttc aca gac tgg cca gag cag ggc gtgccc aag aca ggc gag gga ttc 5785 Phe Thr Asp Trp Pro Glu Gln Gly Val ProLys Thr Gly Glu Gly Phe 1790 1795 1800 1805 att gac ttc atc ggg cag gtgcat aag acc aag gag cag ttt gga cag 5833 Ile Asp Phe Ile Gly Gln Val HisLys Thr Lys Glu Gln Phe Gly Gln 1810 1815 1820 gat ggg cct atc acg gtgcac tgc agt gct ggc gtg ggc cgc acc ggg 5881 Asp Gly Pro Ile Thr Val HisCys Ser Ala Gly Val Gly Arg Thr Gly 1825 1830 1835 gtg ttc atc act ctgagc atc gtc ctg gag cgc atg cgc tat gag ggc 5929 Val Phe Ile Thr Leu SerIle Val Leu Glu Arg Met Arg Tyr Glu Gly 1840 1845 1850 gtg gtc gac atgttt cag acc gtg aag acc ctg cgt aca cag cgt cct 5977 Val Val Asp Met PheGln Thr Val Lys Thr Leu Arg Thr Gln Arg Pro 1855 1860 1865 gcc atg gtgcag aca gag gac cag tat cag ctg tgc tac cgt gcg gcc 6025 Ala Met Val GlnThr Glu Asp Gln Tyr Gln Leu Cys Tyr Arg Ala Ala 1870 1875 1880 1885 ctggag tac ctc ggc agc ttt gac cac tat gca acg taa ctaccgctcc 6074 Leu GluTyr Leu Gly Ser Phe Asp His Tyr Ala Thr 1890 1895 cctctcctcc gccacccccgccgtggggct ccggagggga cccagctcct ctgagccata 6134 ccgaccatcg tccagccctcctacgcagat gctgtcactg gcagagcaca gcccacgggg 6194 atcacagcgt ttcaggaacgttgccacacc aatcagagag cctagaacat ccctgggcaa 6254 gtggatggcc cagcaggcaggcactgtggc ccttctgtcc accagaccca cctggagccc 6314 gcttcaagct ctctgttgcgctcccgcatt tctcatgctt cttctcatgg ggtggggttg 6374 gggcaaagcc tcctttttaatacattaagt ggggtagact gagggatttt agcctcttcc 6434 ctctgatttt tcctttcgcgaatccgtatc tgcagaatgg gccactgtag gggttggggt 6494 ttattttgtt ttgttttttttttttttttg tatgacttct gctgaaggac agaacattgc 6554 cttcctcgtg cagagctggggctgccagcc tgagcggagg ctcggccgtg ggccgggagg 6614 cagtgctgat ccggctgctcctccagccct tcagacgaga tcctgtttca gctaaatgca 6674 gggaaactca atgtttttttaagttttgtt ttccctttaa agcctttttt taggccacat 6734 tgacagtggt gggcggggagaagataggga acactcatcc ctggtcgtct atcccagtgt 6794 gtgtttaaca ttcacagcccagaaccacag atgtgtctgg gagagcctgg caaggcattc 6854 ctcatcacca tcgtgtttgcaaaggttaaa acaaaaacaa aaaaccacaa aaataaaaaa 6914 caaaaaaaac aaaaaacccaaaaaaaaaaa aaaaaagagt cagcccttgg cttctgcttc 6974 aaaccctcaa gaggggaagcaactccgtgt gcctggggtt cccgagggag ctgctggctg 7034 acctgggccc acagagcctggctttggtcc ccagcattgc agtatggtgt ggtgtttgta 7094 ggctgtgggg tctggctgtgtggccaaggt gaatagcaca ggttagggtg tgtgccacac 7154 cccatgcacc tcagggccaagcgggggcgt ggctggcctt tcaggtccag gccagtgggc 7214 ctggtagcac atgtctgtcctcagagcagg ggccagatga ttttcctccc tggtttgcag 7274 ctgttttcaa agcccccgataatcgctctt ttccactcca agatgccctc ataaaccaat 7334 gtggcaagac tactggacttctatcaatgg tactctaatc agtccttatt atcccagctt 7394 gctgaggggc agggagagcgcctcttcctc tgggcagcgc tatctagata ggtaagtggg 7454 ggcggggaag ggtgcatagctgttttagct gagggacgtg gtgccgacgt ccccaaacct 7514 agctaggcta agtcaagatcaacattccag ggttggtaat gttggatgat gaaacattca 7574 tttttacctt gtggatgctagtgctgtaga gttcactgtt gtacacagtc tgttttctat 7634 ttgttaagaa aaactacagcatcattgcat aattcttgat ggtaataaat ttgaataatc 7694 agatttct 7702 5 20 DNAArtificial Sequence PCR Primer 5 catcgccatc ctcttgttca 20 6 20 DNAArtificial Sequence PCR Primer 6 ccgatcgact gctcatcctt 20 7 27 DNAArtificial Sequence PCR Probe 7 aaggaaaagg acccactctc cgtcctc 27 8 19DNA Artificial Sequence PCR Primer 8 gaaggtgaag gtcggagtc 19 9 20 DNAArtificial Sequence PCR Primer 9 gaagatggtg atgggatttc 20 10 20 DNAArtificial Sequence PCR Probe 10 caagcttccc gttctcagcc 20 11 6143 DNAMus musculus CDS (1)...(4435) misc_feature 788, 811, 844, 941-1022,1531, 1534, 1555-1630, 6094 n = A,T,C or G 11 g act ggg ctg tcg gga ggggtg gcc tcc ttc gtg tgc caa gcc aca ggg 49 Thr Gly Leu Ser Gly Gly ValAla Ser Phe Val Cys Gln Ala Thr Gly 1 5 10 15 gaa ccc aag cct cga atcacg tgg atg aag aag ggg aag aaa gtc agc 97 Glu Pro Lys Pro Arg Ile ThrTrp Met Lys Lys Gly Lys Lys Val Ser 20 25 30 tcc cag cgc ttt gag gta attgag ttt gac gat gga gcg ggg tca gtg 145 Ser Gln Arg Phe Glu Val Ile GluPhe Asp Asp Gly Ala Gly Ser Val 35 40 45 ctg cgg atc cag cca tta cga gtgcag cga gac gaa gcc atc tat gag 193 Leu Arg Ile Gln Pro Leu Arg Val GlnArg Asp Glu Ala Ile Tyr Glu 50 55 60 tgc aca gcc acg aac agt ctc ggg gagatc aac aca agt gcc aag ctg 241 Cys Thr Ala Thr Asn Ser Leu Gly Glu IleAsn Thr Ser Ala Lys Leu 65 70 75 80 tca gtg ctt gaa gag gac cag ctg ccgtct ggg ttc ccg act atc gac 289 Ser Val Leu Glu Glu Asp Gln Leu Pro SerGly Phe Pro Thr Ile Asp 85 90 95 atg gga cct cag ctg aag gtg gtg gag aagggt cgc act gcc acc atg 337 Met Gly Pro Gln Leu Lys Val Val Glu Lys GlyArg Thr Ala Thr Met 100 105 110 ctg tgt gca gcc ggt ggg aac cca gac cctgag atc tct tgg ttc aaa 385 Leu Cys Ala Ala Gly Gly Asn Pro Asp Pro GluIle Ser Trp Phe Lys 115 120 125 gac ttc ctt cct gtg gac cct gct gca agcaac ggt cgt atc aaa cag 433 Asp Phe Leu Pro Val Asp Pro Ala Ala Ser AsnGly Arg Ile Lys Gln 130 135 140 ctg cga tca ggt gca ttg cag ata gag agcagc gag gag tct gac caa 481 Leu Arg Ser Gly Ala Leu Gln Ile Glu Ser SerGlu Glu Ser Asp Gln 145 150 155 160 ggc aag tac gag tgt gtg gcc acc aactct gca ggc aca cgc tac tcg 529 Gly Lys Tyr Glu Cys Val Ala Thr Asn SerAla Gly Thr Arg Tyr Ser 165 170 175 gcc ccc gcc aac ctg tat gtg cga gtgcgt cgc gtg gct cct cgt ttc 577 Ala Pro Ala Asn Leu Tyr Val Arg Val ArgArg Val Ala Pro Arg Phe 180 185 190 tcc atc cct ccc agc agc caa gag gtgatg ccc ggc ggc agc gtg aat 625 Ser Ile Pro Pro Ser Ser Gln Glu Val MetPro Gly Gly Ser Val Asn 195 200 205 ctc aca tgt gtg cca gtg gcc gcg cccatg ccg tat gtg aaa tgg atg 673 Leu Thr Cys Val Pro Val Ala Ala Pro MetPro Tyr Val Lys Trp Met 210 215 220 atg gac gcc gag gaa ctg acc aaa gaggat gag atg cca gtc cgc cga 721 Met Asp Ala Glu Glu Leu Thr Lys Glu AspGlu Met Pro Val Arg Arg 225 230 235 240 aat ggt ctg gag ctc agc aat gtcatg cga tct gcc aac tat acc tgt 769 Asn Gly Leu Glu Leu Ser Asn Val MetArg Ser Ala Asn Tyr Thr Cys 245 250 255 gtg gcc atc tct tca tta ngc atgata gaa gcc acg gcc can gtc aca 817 Val Ala Ile Ser Ser Leu Xaa Met IleGlu Ala Thr Ala Xaa Val Thr 260 265 270 gta aaa gct ctg gca aag cct tcaatn gat cct gtg gtg aca gag aca 865 Val Lys Ala Leu Ala Lys Pro Ser XaaAsp Pro Val Val Thr Glu Thr 275 280 285 acc ggc cac agt ggt act ctg acatgg gac tct gga aat acc gag cct 913 Thr Gly His Ser Gly Thr Leu Thr TrpAsp Ser Gly Asn Thr Glu Pro 290 295 300 gtg ctc tac cgc atc aag aac cgcgca nnn nnn nnn nnn nnn nnn nnn 961 Val Leu Tyr Arg Ile Lys Asn Arg AlaXaa Xaa Xaa Xaa Xaa Xaa Xaa 305 310 315 320 nnn nnn nnn nnn nnn nnn nnnnnn nnn nnn nnn nnn nnn nnn nnn nnn 1009 Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 325 330 335 nnn nnn nnn nnn ntc acg cccacc gta gag gcc cgt aca gca cag tcc 1057 Xaa Xaa Xaa Xaa Xaa Thr Pro ThrVal Glu Ala Arg Thr Ala Gln Ser 340 345 350 acc cca tca gcc cct ccc cagaag gtg aca tgt gtg agc acg ggc tcc 1105 Thr Pro Ser Ala Pro Pro Gln LysVal Thr Cys Val Ser Thr Gly Ser 355 360 365 acc acg gtc cgg gta agt tgggtt cca ccg ccg gcc gac agc cgc aac 1153 Thr Thr Val Arg Val Ser Trp ValPro Pro Pro Ala Asp Ser Arg Asn 370 375 380 ggc att atc acc cag tac tccgtg gcc tat gag gca gtg gac ggc gaa 1201 Gly Ile Ile Thr Gln Tyr Ser ValAla Tyr Glu Ala Val Asp Gly Glu 385 390 395 400 gac cgt aag cga cat gtggtg gat ggc atc agc cgt gag cat tcc agc 1249 Asp Arg Lys Arg His Val ValAsp Gly Ile Ser Arg Glu His Ser Ser 405 410 415 tgg gac ctg ctg ggc ctggag aag tgg acg gag tac cgg gtg tgg gtg 1297 Trp Asp Leu Leu Gly Leu GluLys Trp Thr Glu Tyr Arg Val Trp Val 420 425 430 cgg gca cac aca gat gtgggc cct ggc cct gag agc agc ccg gtg ctg 1345 Arg Ala His Thr Asp Val GlyPro Gly Pro Glu Ser Ser Pro Val Leu 435 440 445 gtg cgc acc gat gag gacgtg cct agc ggg cca cca cgg aag gta gag 1393 Val Arg Thr Asp Glu Asp ValPro Ser Gly Pro Pro Arg Lys Val Glu 450 455 460 gtt gag cct ctg aac tccact gct gtg cat gtc tcc tgg aag ctg ccc 1441 Val Glu Pro Leu Asn Ser ThrAla Val His Val Ser Trp Lys Leu Pro 465 470 475 480 gtc ccc aac aag cagcac gga cag att cgt ggc tac cag gtc acc tat 1489 Val Pro Asn Lys Gln HisGly Gln Ile Arg Gly Tyr Gln Val Thr Tyr 485 490 495 gtg cgg ttg gag aatggt gag ccc cga agc caa ccc atc atn ccn gat 1537 Val Arg Leu Glu Asn GlyGlu Pro Arg Ser Gln Pro Ile Xaa Pro Asp 500 505 510 gtc atg ctg gct gaggcn nnn nnn nnn nnn nnn nnn nnn nnn nnn nnn 1585 Val Met Leu Ala Glu AlaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 515 520 525 nnn nnn nnn nnn nnnnnn nnn nnn nnn nnn nnn nnn nnn nnn nnn aca 1633 Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Thr 530 535 540 ctc ttg ggc ctt aaaccg gac acc act ttg aac att aag gtc cgt gca 1681 Leu Leu Gly Leu Lys ProAsp Thr Thr Leu Asn Ile Lys Val Arg Ala 545 550 555 560 cat acc agc aaaggc gcc ggc cct ctc agc ccc agc atc cag tcc cgg 1729 His Thr Ser Lys GlyAla Gly Pro Leu Ser Pro Ser Ile Gln Ser Arg 565 570 575 acc atg ccc gtggag caa gtg ttt gcc aag aat ttc cgt gtg gcc gct 1777 Thr Met Pro Val GluGln Val Phe Ala Lys Asn Phe Arg Val Ala Ala 580 585 590 gcg atg aag acatct gtg ctg ctc agt tgg gag gtc ccc gac tct tat 1825 Ala Met Lys Thr SerVal Leu Leu Ser Trp Glu Val Pro Asp Ser Tyr 595 600 605 aag tca gct gtgccc ttc aag atc ctg tac aat ggg cag agc gtg gag 1873 Lys Ser Ala Val ProPhe Lys Ile Leu Tyr Asn Gly Gln Ser Val Glu 610 615 620 gtg gat ggg cactcg atg cgg aag ctg att gca gac ctg caa ccc aac 1921 Val Asp Gly His SerMet Arg Lys Leu Ile Ala Asp Leu Gln Pro Asn 625 630 635 640 acg gag tactcc ttc gtc ctg atg aat cgt ggc agt agc gcc ggg ggc 1969 Thr Glu Tyr SerPhe Val Leu Met Asn Arg Gly Ser Ser Ala Gly Gly 645 650 655 cta cag cacctg gtg tcc atc cgc act gcc ccg gac ctc cta ccc cag 2017 Leu Gln His LeuVal Ser Ile Arg Thr Ala Pro Asp Leu Leu Pro Gln 660 665 670 aag cca ctgcct gcc tcc gcc ttt ata gag gat ggc cgc ttc tcc ctc 2065 Lys Pro Leu ProAla Ser Ala Phe Ile Glu Asp Gly Arg Phe Ser Leu 675 680 685 tcc atg cctcaa gtg cag gac ccc tcg cta gtc agg tgg ttc tac att 2113 Ser Met Pro GlnVal Gln Asp Pro Ser Leu Val Arg Trp Phe Tyr Ile 690 695 700 gtg gtg gtgccc att gac cgt gtg ggc ggg aac ttg ctg gca cca aga 2161 Val Val Val ProIle Asp Arg Val Gly Gly Asn Leu Leu Ala Pro Arg 705 710 715 720 tgg aacaca cca gag gag ttg gag ctg gac gag ctt ctg gag gcc atc 2209 Trp Asn ThrPro Glu Glu Leu Glu Leu Asp Glu Leu Leu Glu Ala Ile 725 730 735 gag cagggc gag gag aaa cag cgg agg cgc cgg cgc caa gca gag cgg 2257 Glu Gln GlyGlu Glu Lys Gln Arg Arg Arg Arg Arg Gln Ala Glu Arg 740 745 750 ctg aagcct tat gtg gcg gcc caa gtg gat gcg ctc cct gac acc ttc 2305 Leu Lys ProTyr Val Ala Ala Gln Val Asp Ala Leu Pro Asp Thr Phe 755 760 765 acc ctgggg gac aag aag agc tac cgc ggc ttc tac aac cgg ccc ctg 2353 Thr Leu GlyAsp Lys Lys Ser Tyr Arg Gly Phe Tyr Asn Arg Pro Leu 770 775 780 tct ccggat ctg agt tac cag tgc ttc gtt ctc gcc tcc ctc aag gaa 2401 Ser Pro AspLeu Ser Tyr Gln Cys Phe Val Leu Ala Ser Leu Lys Glu 785 790 795 800 cccatg gac cag aag cgc tac gcc tcc agc ccc tac tcg gac gag att 2449 Pro MetAsp Gln Lys Arg Tyr Ala Ser Ser Pro Tyr Ser Asp Glu Ile 805 810 815 gtagtc cag gtg acg cca gca cag cag cag gag gag ccc gag atg ctg 2497 Val ValGln Val Thr Pro Ala Gln Gln Gln Glu Glu Pro Glu Met Leu 820 825 830 tgggtg aca ggc cct gtc ctg gcg gtc att ctc atc ata ctc att gtc 2545 Trp ValThr Gly Pro Val Leu Ala Val Ile Leu Ile Ile Leu Ile Val 835 840 845 atcgcc atc ctc ctg ttc aag agg aag aga aca cac tcc cca tca tca 2593 Ile AlaIle Leu Leu Phe Lys Arg Lys Arg Thr His Ser Pro Ser Ser 850 855 860 aaggat gag cag tca atc ggg ctg aag gac tcc ctg ttg gcc cac tct 2641 Lys AspGlu Gln Ser Ile Gly Leu Lys Asp Ser Leu Leu Ala His Ser 865 870 875 880tct gac cct gtg gag atg cga agg ctt aac tac cag acc cca ggt atg 2689 SerAsp Pro Val Glu Met Arg Arg Leu Asn Tyr Gln Thr Pro Gly Met 885 890 895cga gac cac ccg ccc atc ccc atc act gac ctg gca gac aat att gag 2737 ArgAsp His Pro Pro Ile Pro Ile Thr Asp Leu Ala Asp Asn Ile Glu 900 905 910cgc ctc aaa gcc aac gat ggg ctc aag ttc tcc cag gag tat gag tcc 2785 ArgLeu Lys Ala Asn Asp Gly Leu Lys Phe Ser Gln Glu Tyr Glu Ser 915 920 925att gac cct gga cag cag ttc aca tgg gag aat tcc aac tcg gag gtg 2833 IleAsp Pro Gly Gln Gln Phe Thr Trp Glu Asn Ser Asn Ser Glu Val 930 935 940aac aag ccc aag aac cgc tat gca aat gtc att gcc tat gac cat tct 2881 AsnLys Pro Lys Asn Arg Tyr Ala Asn Val Ile Ala Tyr Asp His Ser 945 950 955960 cga gtc ctc ctc acc tcc att gat ggt gtt cct ggg agt gac tac atc 2929Arg Val Leu Leu Thr Ser Ile Asp Gly Val Pro Gly Ser Asp Tyr Ile 965 970975 aat gcc aac tac att gat ggc tac cga aag cag aat gcc tac atc gcc 2977Asn Ala Asn Tyr Ile Asp Gly Tyr Arg Lys Gln Asn Ala Tyr Ile Ala 980 985990 aca caa ggt ccg ctg ccc gag acc atg ggc gat ttc tgg agg atg gtg 3025Thr Gln Gly Pro Leu Pro Glu Thr Met Gly Asp Phe Trp Arg Met Val 995 10001005 tgg gaa cag cgc aca gcc aca gtg gtc atg atg acc agg cta gag gag3073 Trp Glu Gln Arg Thr Ala Thr Val Val Met Met Thr Arg Leu Glu Glu1010 1015 1020 aaa tcc cgg gtg aag tgt gat cag tat tgg cca gtc cgt ggcact gag 3121 Lys Ser Arg Val Lys Cys Asp Gln Tyr Trp Pro Val Arg Gly ThrGlu 1025 1030 1035 1040 acc tat ggc ctc att cag gtg acc ctg gtg gac actgtg gag ttg gcc 3169 Thr Tyr Gly Leu Ile Gln Val Thr Leu Val Asp Thr ValGlu Leu Ala 1045 1050 1055 aca tac acc atg cgc acc ttt gcc ctc cat aagagt ggc tcc agt gag 3217 Thr Tyr Thr Met Arg Thr Phe Ala Leu His Lys SerGly Ser Ser Glu 1060 1065 1070 aag cgt gag ctg cgt cag ttc cag ttc atggcc tgg cca gac cac ggc 3265 Lys Arg Glu Leu Arg Gln Phe Gln Phe Met AlaTrp Pro Asp His Gly 1075 1080 1085 gtt cct gag tac ccc act ccc atc ttggcc ttc ctg aga cgg gtc aag 3313 Val Pro Glu Tyr Pro Thr Pro Ile Leu AlaPhe Leu Arg Arg Val Lys 1090 1095 1100 gcc tgt aac cca cta gat gcg gggccc atg gtg gtg cat tgc agt gcg 3361 Ala Cys Asn Pro Leu Asp Ala Gly ProMet Val Val His Cys Ser Ala 1105 1110 1115 1120 ggt gtg ggg cgc aca ggctgc ttc atc gtc atc gac gca atg ctg gag 3409 Gly Val Gly Arg Thr Gly CysPhe Ile Val Ile Asp Ala Met Leu Glu 1125 1130 1135 cgt atg aag cac gagaag acg gtt gac atc tat ggc cac gtg acg tgc 3457 Arg Met Lys His Glu LysThr Val Asp Ile Tyr Gly His Val Thr Cys 1140 1145 1150 atg cgc tca caaagg aac tac atg gtg cag acc gag gac cag tat gtg 3505 Met Arg Ser Gln ArgAsn Tyr Met Val Gln Thr Glu Asp Gln Tyr Val 1155 1160 1165 ttc atc cacgag gcc ctg cta gag gct gcc atg tgc gga cac acc gag 3553 Phe Ile His GluAla Leu Leu Glu Ala Ala Met Cys Gly His Thr Glu 1170 1175 1180 gtg ctcgct cgg aac ctc tat gcc cac atc cag aag cta ggc caa gtg 3601 Val Leu AlaArg Asn Leu Tyr Ala His Ile Gln Lys Leu Gly Gln Val 1185 1190 1195 1200cct ccc ggg gag agc gtc acg gcc atg gaa ctt gag ttc aag ttg ctg 3649 ProPro Gly Glu Ser Val Thr Ala Met Glu Leu Glu Phe Lys Leu Leu 1205 12101215 gcc aac tcc aag gcc cac acg tcg cgc ttt gtc agt gcc aac ctg ccc3697 Ala Asn Ser Lys Ala His Thr Ser Arg Phe Val Ser Ala Asn Leu Pro1220 1225 1230 tgc aac aag ttc aag aac cgc cta gtg aac atc atg ccc tatgag ctg 3745 Cys Asn Lys Phe Lys Asn Arg Leu Val Asn Ile Met Pro Tyr GluLeu 1235 1240 1245 acc cga gtg tgc ttg caa ccc atc cgt ggt gtg gag ggctca gac tac 3793 Thr Arg Val Cys Leu Gln Pro Ile Arg Gly Val Glu Gly SerAsp Tyr 1250 1255 1260 atc aat gcc agc ttt cta gat ggc tac aga cag cagaag gcc tac ata 3841 Ile Asn Ala Ser Phe Leu Asp Gly Tyr Arg Gln Gln LysAla Tyr Ile 1265 1270 1275 1280 gct aca cag ggg cct ctg gca gag agc accgag gac ttc tgg cgc atg 3889 Ala Thr Gln Gly Pro Leu Ala Glu Ser Thr GluAsp Phe Trp Arg Met 1285 1290 1295 tta tgg gag cac aat tcc acc atc atcgtc atg ctg acc aag ctt cgg 3937 Leu Trp Glu His Asn Ser Thr Ile Ile ValMet Leu Thr Lys Leu Arg 1300 1305 1310 gag atg ggc agg gag aaa tgt caccag tac tgg cca gca gag cgc tcc 3985 Glu Met Gly Arg Glu Lys Cys His GlnTyr Trp Pro Ala Glu Arg Ser 1315 1320 1325 gct cgc tat cag tac ttc gttgtt gac ccg atg gct gag tac aac atg 4033 Ala Arg Tyr Gln Tyr Phe Val ValAsp Pro Met Ala Glu Tyr Asn Met 1330 1335 1340 ccc cag tat att ctg cgtgaa ttc aaa gtc aca gac gcc cgg gat ggg 4081 Pro Gln Tyr Ile Leu Arg GluPhe Lys Val Thr Asp Ala Arg Asp Gly 1345 1350 1355 1360 cag tca agg acaatc cga cag ttc cag ttt aca gac tgg cca gag caa 4129 Gln Ser Arg Thr IleArg Gln Phe Gln Phe Thr Asp Trp Pro Glu Gln 1365 1370 1375 gga gta cccaaa aca ggt gaa ggc ttc atc gac ttc atc ggg cag gtg 4177 Gly Val Pro LysThr Gly Glu Gly Phe Ile Asp Phe Ile Gly Gln Val 1380 1385 1390 cac aagaca aag gag cag ttt ggc cag gat ggg ccc atc acg gtg cac 4225 His Lys ThrLys Glu Gln Phe Gly Gln Asp Gly Pro Ile Thr Val His 1395 1400 1405 tgcagt gct ggt gtg ggc cgc acc ggt gtg ttc atc acc ctg agc att 4273 Cys SerAla Gly Val Gly Arg Thr Gly Val Phe Ile Thr Leu Ser Ile 1410 1415 1420gtc ctg gag cgc atg cgc tat gag ggt gtg gtt gac atg ttc cag acc 4321 ValLeu Glu Arg Met Arg Tyr Glu Gly Val Val Asp Met Phe Gln Thr 1425 14301435 1440 gtg aag acc ctc cgc aca cag cgc cct gca atg gtg cag aca gaggac 4369 Val Lys Thr Leu Arg Thr Gln Arg Pro Ala Met Val Gln Thr Glu Asp1445 1450 1455 caa tac cag ctg tgc tac cgt gcg gcc ctg gaa tac ctc ggcagc ttt 4417 Gln Tyr Gln Leu Cys Tyr Arg Ala Ala Leu Glu Tyr Leu Gly SerPhe 1460 1465 1470 gat cac tat gca acg taa ctactgctcc cctctcctccgacgctcccc 4465 Asp His Tyr Ala Thr * 1475 cgcggctccg gagggacccagctcctctga gccataccaa ccatcgtcca gccctcctgc 4525 acggatgctg ttgccggcagagcacagccc actgggatca cagcatttcg gggaacattg 4585 ccacaccagt cagagagcccagaacacctg ggcaagtagg cggactggca gcctggctct 4645 gggccctcgt ccaccgggccaagtggagcc ccgcttcaag ctctctgttc agctccgcgt 4705 tctcatgctt ctcatggggtgggaaaaggg ggcaaagccc ccacttttta tacactaggc 4765 ggggtagact gcggggtcctagcctcttcc tccgactttg cttttgcagg tctttcactg 4825 cagatggggc tgctgtgggagttgggactt gtttgttttc ctttttgagt tcacgttgga 4885 tcctttcttg tacaacttctgcggaaggac acagtagtaa ctcgccttcc ttgtgcagag 4945 ctagggccct acctgagcaagtcggctgtg gcccgggagg cagcgtgact cctgctgtcc 5005 tccagccttt cagatgagatcctattccag ccaaatgcag ggaaacactt tattttgttt 5065 gttttaggtt ttgtttttccttgagagcct ttttttaggc cccacagaca gtggtgggtg 5125 gggaggcgat aggaaacacattccccagtg tgcatttaac attcatagcc tacaaccaca 5185 gacgtgtctg ggggagcctggcaaggcgtt cctcgtcacc atcgtgtttg caaaggttca 5245 aaaaacaaaa atcaaaaaaaaaaaaaccat aaaaatattt ttttttaaga aaagaaataa 5305 agattcatcc cctggcctctacttcagatt cgaagtggga ggcaactcaa tgtgccctgg 5365 ggctcgccat gggcccacagagtctcgcta tcatccccag cgtcgcagtg tggcaggtgt 5425 ttgtaggctg tgggttctggccacctcggg aaatgaatgg cacaggtgag ggcctgtgcc 5485 acgccccaca cacctcagggccaagcgggg gcgtggctgg cccttcaggt caggccagtg 5545 ggcctggtag cacatgtctgtcctcagccg gacagatgct ttctctcctg gtttgcagct 5605 gtcttcaaat ccccccatgacccgctcttc ccactccttc aagttgccct cacaaaccaa 5665 tgtggcaaga ctactggacttgtatccatg gtactatact cagtcctctt atctcagctt 5725 gctgaggggc agggagagggtctcttcctc tgggcagcac tatctagata ggtaagtggg 5785 ggcggggaag ggtgcatagctgttttagcc gagggactcg ataccgacgt ccccagatat 5845 agctaggcta agtcaagatcaacagtccgg ggttggggtg tggatgaaac attcattttt 5905 accttgtgga tgctagtgctgtagagttca ctgtggtaca cagtccgttt tctatttgtt 5965 aagaaaaact acagcgatcatgtgcatact tctgtgatgg tgataaattt gaataatcca 6025 gattcttaca cactagcctctgtctcagct ctgtatctag agtgggatct taagtctatg 6085 ggggggggnc ccgggggtcctcccaagggg gtgagccccc ctggggggtg cccgacgg 6143 12 19 DNA ArtificialSequence PCR Primer 12 ctcctgcacg gatgctgtt 19 13 20 DNA ArtificialSequence PCR Primer 13 gttccccgaa atgctgtgat 20 14 21 DNA ArtificialSequence PCR Probe 14 cggcagagca cagcccactg g 21 15 20 DNA ArtificialSequence PCR Primer 15 ggcaaattca acggcacagt 20 16 20 DNA ArtificialSequence PCR Primer 16 gggtctcgct cctggaagat 20 17 27 DNA ArtificialSequence PCR Probe 17 aaggccgaga atgggaagct tgtcatc 27 18 7724 DNA H.sapiens 18 cgggagcggc gggagcggtg gcggcggcag aggcggcggc tccagcttcggctccggctc 60 gggctcgggc tccggctccg gctccggctc cggctccagc tcgggtggcggtggcgggag 120 cgggaccagg tggaggcggc ggcggcagag gagtgggagc agcggccctagcggcttgcg 180 gggggacatg cggaccgacg gcccctggat aggcggaagg agtggaggccctggtgcccg 240 gcccttggtg ctgagtatcc agcaagagtg accggggtga agaagcaaagactcggttga 300 ttgtcctggg ctgtggctgg ctgtggagct agagccctgg atggcccctgagccagcccc 360 agggaggacg atggtgcccc ttgtgcctgc actggtgatg cttggtttggtggcaggcgc 420 ccatggtgac agcaaacctg tcttcattaa agtccctgag gaccagactgggctgtcagg 480 aggggtagcc tccttcgtgt gccaagctac aggagaaccc aagccgcgcatcacatggat 540 gaagaagggg aagaaagtca gctcccagcg cttcgaggtc attgagtttgatgatggggc 600 agggtcagtg cttcggatcc agccattgcg ggtgcagcga gatgaagccatctatgagtg 660 tacagctact aacagcctgg gtgagatcaa cactagtgcc aagctctcagtgctcgaaga 720 ggaacagctg ccccctgggt tcccttccat cgacatgggg cctcagctgaaggtggtgga 780 gaaggcacgc acagccacca tgctatgtgc cgcaggcgga aatccagaccctgagatttc 840 ttggttcaag gacttccttc ctgtagaccc tgccacgagc aacggccgcatcaagcagct 900 gcgttcaggt gccttgcaga tagagagcag tgaggaatcc gaccaaggcaagtacgagtg 960 tgtggcgacc aactcggcag gcacacgtta ctcagcccct gcgaacctgtatgtgcgagt 1020 gcgccgcgtg gctcctcgtt tctccatccc tcccagcagc caggaggtgatgccaggcgg 1080 cagcgtgaac ctgacatgcg tggcagtggg tgcacccatg ccctacgtgaagtggatgat 1140 gggggccgag gagctcacca aggaggatga gatgccagtt ggccgcaacgtcctggagct 1200 cagcaatgtc gtacgctctg ccaactacac ctgtgtggcc atctcctcgctgggcatgat 1260 cgaggccaca gcccaggtca cagtgaaagc tcttccaaag cctccgattgatcttgtggt 1320 gacagagaca actgccacca gtgtcaccct cacctgggac tctgggaactcggagcctgt 1380 aacctactat ggcatccagt accgcgcagc gggcacggag ggcccctttcaggaggtgga 1440 tggtgtggcc accacccgct acagcattgg cggcctcagc cctttctcggaatatgcctt 1500 ccgcgtgctg gcggtgaaca gcatcgggcg agggccgccc agcgaggcagtgcgggcacg 1560 cacgggagaa caggcgccct ccagcccacc gcgccgcgtg caggcacgcatgctgagcgc 1620 cagcaccatg ctggtgcagt gggagcctcc cgaggagccc aacggcctggtgcggggata 1680 ccgcgtctac tatactccgg actcccgccg ccccccgaac gcctggcacaagcacaacac 1740 cgacgcgggg ctcctcacga ccgtgggcag cctgctgcct ggcatcacctacagcctgcg 1800 cgtgcttgcc ttcaccgccg tgggcgatgg ccctcccagc cccaccatccaggtcaagac 1860 gcagcaggga gtgcctgccc agcccgcgga cttccaggcc gaggtggagtcggacaccag 1920 gatccagctc tcgtggctgc tgccccctca ggagcggatc atcatgtatgaactggtgta 1980 ctgggcggca gaggacgaag accaacagca caaggtcacc ttcgacccaacctcctccta 2040 cacactagag gacctgaagc ctgacacact ctaccgcttc cagctggctgcacgctcgga 2100 tatgggggtg ggcgtcttca cccccaccat tgaggcccgc acagcccagtccaccccctc 2160 cgcccctccc cagaaggtga tgtgtgtgag catgggctcc accacggtccgggtaagttg 2220 ggtcccgccg cctgccgaca gccgcaacgg cgttatcacc cagtactccgtggcccacga 2280 ggcggtggac ggcgaggacc gcgggcggca tgtggtggat ggcatcagccgtgagcactc 2340 cagctgggac ctggtgggcc tggagaagtg gacggagtac cgggtgtgggtgcgggcaca 2400 cacagacgtg ggccccggcc ccgagagcag cccggtgctg gtgcgcaccgatgaggacgt 2460 gcccagcggg cctccgcgga aggtggaggt ggagccactg aactccactgctgtgcatgt 2520 ctactggaag ctgcctgtcc ccagcaagca gcatggccag atccgcggctaccaggtcac 2580 ctacgtgcgg ctggagaatg gcgagccccg tggactcccc atcatccaagacgtcatgct 2640 agccgaggcc cagtggcggc cagaggagtc cgaggactat gaaaccactatcagcggcct 2700 gaccccggag accacctact ccgttactgt tgctgcctat accaccaagggggatggtgc 2760 ccgcagcaag cccaaaattg tcactacaac aggtgcagtc ccaggccggcccaccatgat 2820 gatcagcacc acggccatga acactgcgct gctccagtgg cacccacccaaggaactgcc 2880 tggcgagctg ctgggctacc ggctgcagta ctgccgggcc gacgaggcgcggcccaacac 2940 catagatttc ggcaaggatg accagcactt cacagtcacc ggcctgcacaaggggaccac 3000 ctacatcttc cggcttgctg ccaagaaccg ggctggcttg ggtgaggagttcgagaagga 3060 gatcaggacc cccgaggacc tgcccagcgg cttcccccaa aacctgcatgtgacaggact 3120 gaccacgtct accacagaac tggcctggga cccgccagtg ctggcggagaggaacgggcg 3180 catcatcagc tacaccgtgg tgttccgaga catcaacagc caacaggagctgcagaacat 3240 cacgacagac acccgcttta cccttactgg cctcaagcca gacaccacttacgacatcaa 3300 ggtccgcgca tggaccagca aaggctctgg cccactcagc cccagcatccagtcccggac 3360 catgccggtg gagcaagtgt ttgccaagaa cttccgggtg gcggctgcaatgaagacgtc 3420 tgtgctgctc agctgggagg ttcccgactc ctataagtca gctgtgccctttaagattct 3480 gtacaatggg cagagtgtgg aggtggacgg gcactcgatg cggaagctgatcgcagacct 3540 gcagcccaac acagagtact cgtttgtgct gatgaaccgt ggcagcagcgcagggggcct 3600 gcagcacctg gtgtccatcc gcacagcccc cgacctcctg cctcacaagccgctgcctgc 3660 ctctgcctac atagaggacg gccgcttcga tctctccatg ccccatgtgcaagacccctc 3720 gcttgtcagg tggttctaca ttgttgtggt acccattgac cgtgtgggcgggagcatgct 3780 gacgccaagg tggagcacac ccgaggaact ggagctggac gagcttctagaagccatcga 3840 gcaaggcgga gaggagcagc ggcggcggcg gcggcaggca gaacgtctgaagccatatgt 3900 ggctgctcaa ctggatgtgc tcccggagac ctttaccttg ggggacaagaagaactaccg 3960 gggcttctac aaccggcccc tgtctccgga cttgagctac cagtgctttgtgcttgcctc 4020 cttgaaggaa cccatggacc agaagcgcta tgcctccagc ccctactcggatgagatcgt 4080 ggtccaggtg acaccagccc agcagcagga ggagccggag atgctgtgggtgacgggtcc 4140 cgtgctggca gtcatcctca tcatcctcat tgtcatcgcc atcctcttgttcaaaaggaa 4200 aaggacccac tctccgtcct ctaaggatga gcagtcgatc ggactgaaggactccttgct 4260 ggcccactcc tctgaccctg tggagatgcg gaggctcaac taccagaccccaggtatgcg 4320 agaccaccca cccatcccca tcaccgacct ggcggacaac atcgagcgcctcaaagccaa 4380 cgatggcctc aagttctccc aggagtatga gtccatcgac cctggacagcagttcacgtg 4440 ggagaattca aacctggagg tgaacaagcc caagaaccgc tatgcgaatgtcatcgccta 4500 cgaccactct cgagtcatcc ttacctctat cgatggcgtc cccgggagtgactacatcaa 4560 tgccaactac atcgatggct accgcaagca gaatgcctac atcgccacgcagggccccct 4620 gcccgagacc atgggcgatt tctggagaat ggtgtgggaa cagcgcacggccactgtggt 4680 catgatgaca cggctggagg agaagtcccg ggtaaaatgt gatcagtactggccagcccg 4740 tggcaccgag acctgtggcc ttattcaggt gaccctgttg gacacagtggagctggccac 4800 atacactgtg cgcaccttcg cactccacaa gagtggctcc agtgagaagcgtgagctgcg 4860 tcagtttcag ttcatggcct ggccagacca tggagttcct gagtacccaactcccatcct 4920 ggccttccta cgacgggtca aggcctgcaa ccccctagac gcagggcccatggtggtgca 4980 ctgcagcgcg ggcgtgggcc gcaccggctg cttcatcgtg attgatgccatgttggagcg 5040 gatgaagcac gagaagacgg tggacatcta tggccacgtg acctgcatgcgatcacagag 5100 gaactacatg gtgcagacgg aggaccagta cgtgttcatc catgaggcgctgctggaggc 5160 tgccacgtgc ggccacacag aggtgcctgc ccgcaacctg tatgcccacatccagaagct 5220 gggccaagtg cctccagggg agagtgtgac cgccatggag ctcgagttcaagttgctggc 5280 cagctccaag gcccacacgt cccgcttcat cagcgccaac ctgccctgcaacaagttcaa 5340 gaaccggctg gtgaacatca tgccctacga attgacccgt gtgtgtctgcagcccatccg 5400 tggtgtggag ggctctgact acatcaatgc cagcttcctg gatggttatagacagcagaa 5460 ggcctacata gctacacagg ggcctctggc agagagcacc gaggacttctggcgcatgct 5520 atgggagcac aattccacca tcatcgtcat gctgaccaag cttcgggagatgggcaggga 5580 gaaatgccac cagtactggc cagcagagcg ctctgctcgc taccagtactttgttgttga 5640 cccgatggct gagtacaaca tgccccagta tatcctgcgt gagttcaaggtcacggatgc 5700 ccgggatggg cagtcaagga caatccggca gttccagttc acagactggccagagcaggg 5760 cgtgcccaag acaggcgagg gattcattga cttcatcggg caggtgcataagaccaagga 5820 gcagtttgga caggatgggc ctatcacggt gcactgcagt gctggcgtgggccgcaccgg 5880 ggtgttcatc actctgagca tcgtcctgga gcgcatgcgc tatgagggcgtggtcgacat 5940 gtttcagacc gtgaagaccc tgcgtacaca gcgtcctgcc atggtgcagacagaggacca 6000 gtatcagctg tgctaccgtg cggccctgga gtacctcggc agctttgaccactatgcaac 6060 gtaactaccg ctcccctctc ctccgccacc cccgccgtgg ggctccggaggggacccagc 6120 tcctctgagc cataccgacc atcgtccagc cctcctacgc agatgctgtcactggcagag 6180 cacagcccac ggggatcaca gcgtttcagg aacgttgcca caccaatcagagagcctaga 6240 acatccctgg gcaagtggat ggcccagcag gcaggcactg tggcccttctgtccaccaga 6300 cccacctgga gcccgcttca agctctctgt tgcgctcccg catttctcatgcttcttctc 6360 atggggtggg gttggggcaa agcctccttt ttaatacatt aagtggggtagactgaggga 6420 ttttagcctc ttccctctga tttttccttt cgcgaatccg tatctgcagaatgggccact 6480 gtaggggttg gggtttattt tgttttgttt ttttttttct tgagttcactttggatcctt 6540 attttgtatg acttctgctg aaggacagaa cattgccttc ctcgtgcagagctggggctg 6600 ccagcctgag cggaggctcg gccgtgggcc gggaggcagt gctgatccggctgctcctcc 6660 agcccttcag acgagatcct gtttcagcta aatgcaggga aactcaatgtttttttaagt 6720 tttgttttcc ctttaaagcc tttttttagg ccacattgac agtggtgggcggggagaaga 6780 tagggaacac tcatccctgg tcgtctatcc cagtgtgtgt ttaacattcacagcccagaa 6840 ccacagatgt gtctgggaga gcctggcaag gcattcctca tcaccatcgtgtttgcaaag 6900 gttaaaacaa aaacaaaaaa ccacaaaaat aaaaaacaaa aaaaacaaaaaacccaaaaa 6960 aaaaaaaaaa aagagtcagc ccttggcttc tgcttcaaac cctcaagaggggaagcaact 7020 ccgtgtgcct ggggttcccg agggagctgc tggctgacct gggcccacagagcctggctt 7080 tggtccccag cattgcagta tggtgtggtg tttgtaggct gtggggtctggctgtgtggc 7140 caaggtgaat agcacaggtt agggtgtgtg ccacacccca tgcacctcagggccaagcgg 7200 gggcgtggct ggcctttcag gtccaggcca gtgggcctgg tagcacatgtctgtcctcag 7260 agcaggggcc agatgatttt cctccctggt ttgcagctgt tttcaaagcccccgataatc 7320 gctcttttcc actccaagat gccctcataa accaatgtgg caagactactggacttctat 7380 caatggtact ctaatcagtc cttattatcc cagcttgctg aggggcagggagagcgcctc 7440 ttcctctggg cagcgctatc tagataggta agtgggggcg gggaagggtgcatagctgtt 7500 ttagctgagg gacgtggtgc cgacgtcccc aaacctagct aggctaagtcaagatcaaca 7560 ttccagggtt ggtaatgttg gatgatgaaa cattcatttt taccttgtggatgctagtgc 7620 tgtagagttc actgttgtac acagtctgtt ttctatttgt taagaaaaactacagcatca 7680 ttgcataatt cttgatggta ataaatttga ataatcagat ttct 7724 19813 DNA H. sapiens 19 caccggtctg cccagcagag cgctctgctc gctaccagtacttgttgtga ccccgatggc 60 tgagtacaca tgccccagta tatccgcgtg agttcaaggtcacggatgcc cgggatgggc 120 agtcaaggac aatccggcag ttcacagttc acagactggccagaagcagg gcgtgccaca 180 agacaggcga gggattcact gacttcatcg ggcaggtgcataagaccaag gagcagtttg 240 gacaggatgg gcctatcacg gtgcactgca gtgctggcgtgggccgcacc ggggtgttca 300 tcactctgag catcgtcctg gagcgcatgc gctatgagggcgtggctggc gtttcaggtc 360 caggccagtg ggcctggtag cacatgtctg tcctcagagcaggggccaga tgattttcct 420 ccctggtttg cagctgtttt caaagccccc gataatcgctcttttccact ccaagatgcc 480 ctcataaacc aatgtggcaa gactactgga cttctatcaatggtactcta atcagtcctt 540 attatcccag cttgctgagg ggcagggaga gcgcctcttcctctgggcag cgctatctag 600 ataggtaagt gggggcgggg aagggtgcat agctgttttagctgagggac gtggtgccga 660 cgtccccaaa cctagctagg ctaagtcaag atcaacattccagggttggt aatgttggat 720 gatgaaacat tcatttttac cttgtggatg ctagtgctgtagagttcact gttgtacaca 780 gtctgttttc tattttaaca aggaaactac agc 813 2094001 DNA Homo sapiens misc_feature 605-704, 63461-63560, 92457 n =A,T,C or G 20 aatggggtcc tagagtggta ttaggttcat gctagggctg ggagccactagaaggatctg 60 gatgtagtat cagagtttgg tctgtctatg ttcattattg gggttcgggttcaatattga 120 ggttccggtc ggtttcgact tggcgttggg ttgtggcctg tcagggcctcagcggctccg 180 cggctctacg agcgagagtg cacgagggga ggggcgccgc cgggggcgcgcacggcaggg 240 gcaggggcgc gggcgcgagc gcgaggggag cgcgcggctg gagctggcgcgggagcggcg 300 ggagcggtgg cggcggcaga ggcggcggct ccagcttcgg ctccggctcgggctcgggct 360 ccggctccgg ctccggctcc ggctccagct cgggtggcgg tggcgggagcgggaccaggt 420 ggagtcggcg gcggcagagg agtgggagca gcggccctag cggcttgcgggggtacatgc 480 ggaccgacgg cccctggata ggcggtgagt gaccccccgg ccccccaccagccccctccg 540 ctcccgtccc ttccccgctc tccttgccct ccccgctagt ccacccggcggagctggggg 600 cggtnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn 660 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnaaccgggtgcaacgcc 720 ctggaggccc tggagcgacc tgtcccgtga ggacgtggcc actcggaccctctccgaaag 780 ttcactcagt ggccgccccg cgtcccgtcc cgtcccgtcc cggctccccatcgccgtgcc 840 ggcgtctctg tctcggctgc tttctgtttt cctcggcgtc tctgcctccccgtgtcagtg 900 cctcccaatc tcacgcccct gcaatcccag ggtctctcca gctgtctccattctttttcc 960 ccaggtcact ctgttctttc tccccaggtc tctcgattct gtctccctgggtcgtcttgt 1020 tccctgtccc tgagtctgtt ctctccgctt gggtctctct atttcctctccctgggtctc 1080 tgtctccctc cttaagtctc tgcctcttgt ctctcaccca tctcttggtgtctctgcact 1140 ctctgtgcta ggcgcccccc catttccctg gaatgctgcc cctctggttcttccaccccg 1200 gagggggagg ggctagactt tctccccatt tcaagccccc ctgccccccagtgcacggcc 1260 cctgagttgg agcaggttgg gggtggggag cgctacctgc ctgagctgtgaaatgggaga 1320 gggaggctgc cccagcctgg gggttgtctg gatctccctg aggggccacagggcagggcc 1380 gggaggctgg attaggttga ggaaggctcc acttttgaag gaacaggggcagggatccag 1440 agcgatctgg tgtcggatct ggcctgagaa gcagggaagt gcactggggtgagaggcgta 1500 gaaaaggatg tggggtggtg gttccaggcg ggtgctcatg gggaggaatctgctgagaag 1560 ggaagatctg gtggggaatc ctttgggagg cttcggggat ctgagggatccagaagagag 1620 ttctgacagg ctatctaagg aaatttctgt gcttagctcc ttattaactcttgcttcagg 1680 gagcatgaaa atccctgttt ttttactgat tatatgaatg agcctagggcttcaagaagg 1740 agcaggtgta actccccaag tacaaccccc tacattctca gccccaggcacaggccacac 1800 tttctcgtac cctcttgcct tcatttccac actcagctca ccccctccacccccacacac 1860 actccctatt cagctgcttt cttgccccct ctcgcatacc tgggcccgcatgccggccag 1920 ggctcaagaa atctgtgcct gcacactctc gcagcatacc tgtgcacatgcacagtcatc 1980 cttgtgtatt tttgtatatg cactttcttg cacattttac ttgctcagtctcttatttga 2040 aatgtcttta tagtgttttt tttagtttcc tttatagttt ccttttccagctgctcttcc 2100 tcttccctct ttctttcctt taacaagaaa attgttttaa cataattccttccttccttc 2160 ctccctttct tccttcttct tgcaactact gatacctccc ctttatccagctttgtgtgc 2220 cctgggcggg ttggcttggt gtacaaggct gccaagagag gagggggtggttgggtgttg 2280 gtgaaggaat ttggcggggc agagttagac tgcagcaaac ggaaactagctacacatctt 2340 ttactgggaa gtgtatggat acgcagcaaa atactagccc tggcagtttgggcacagtcc 2400 accctttttt ccatcagcct gaactatggg agtctagctt tgagggtcctccctgggggc 2460 ggggaggagg ggacccagac ctcctgcagt agaggggttg tgatacacaccacggctcct 2520 ccactttcca gcttcaggcc tcaaagaggc ccctttctcc ctgagtctcaatttcactcc 2580 ctgtaaaatg gggtagctaa tccaacccgg actgggtccc agggctgttgtaagaatcca 2640 agggaactgt gtggcagtgc atcgtcagca gtggcagtca cttcccttgtgactgacact 2700 gctgtgtgct tggttccagc ttgcagtcag ggccctaccc tcgagaagctcgtggtcctt 2760 gggggagcag gttgcaccat gggattgcaa aagcaattca gttacctgtcattgattgct 2820 gactgtgtgc cagaccccgg ggtaggcgct ttcggccccg gctgcctttcttcctttggg 2880 actaacagct atgtagaaga ggcagcccac ccctaaggtt caggcatctgctcaactaaa 2940 gcagctgcgg ttctgagttg ggttagaaca ttgcaagagt tcatcaagagacgggggaga 3000 gttcggaggt cagggtgagg tgagtcccag aggagaccaa gaagaagtgatgtgaactag 3060 gtttggaggg atgagtagga gtttgaagaa gagagaaagg acttgagatagataaaaacc 3120 agtatgaata aagactggga ggtgttgtca cttgtgaagt attcagagatgtggcagatc 3180 acagagggca ttgaatgccc tgttaagtac ctgtaatttt actctgagaacccagagaga 3240 aaacaccaaa ggtttaaagc ttggataggg agggtggagt gtgctgtggtcaggtttatg 3300 tttccagaaa atgctctgtg gctatgggaa aaggatggag acaggatgcccagtggggca 3360 agagggtgca ggtaagtaag gtgagagaag atgctggctt tgataggtggtagcatggtg 3420 ccatcttgaa ggtacacttg tcaggacaga atgatttatt ggatgtgggagtgaaggaga 3480 ggaagaggaa ggagtccagg ttcctcggag tggttgagcg ggccttcactgaaggccctg 3540 aagcttcact gtgttaggga cttggtgagg aggggcagag aataagctcttagttggaca 3600 tgtggccctt gaggtacttg ggacattgga ggctcagtgg gggaggcacatcaaggaggg 3660 gctttggtgt gagggtatgg gagagaaatg tcggtcggac caagagaagcgagagctgta 3720 tgagagacca tagagctgtg cacctcgaga gaaagtaacc tccgacagtggtgaagagct 3780 cgagttttat actgctagga ttggactctg tttactagca gcgtgactttaagcaagtgg 3840 cttaacctct ctgaatccgc ttcctcattt gtcaaatgat tataataaaaacccgtgcct 3900 tataagggtt gttatgagga ttaaagatag tatgcataaa gtgtgtgtagcacagggctt 3960 ggcatctagt gagtactgtt gtctgtgttt gcagttactg taatcatcctcaactgggat 4020 ggtgtaaacc caccctccca tcaggggtca catgaggttg cctttaatgagaagatagac 4080 tgctaaggtt agggtccctg tctgcctaag gttgctgcaa ttctgagctgagattagcag 4140 gtcaaggagc tcaacaaagg gggtgcctgc ccctattgtt atcgatgggattgtttgtgt 4200 tgctcaggtg tttgaccttc aggtgggtgg gcttgagaag actagaggtgaggcctgcag 4260 aagagactcc atgtttgggg taggtggaag ggaattttga gaaggggcagccttggggta 4320 ttgagttctc catttcctcc caccctccct gggccctggg cttctactgaggctctttct 4380 gccaccatct gcctctgtgg tccagatgca ccatgtctct gtgtgaccgtgacttctagg 4440 tctgagaact ctggttttag ctccttagca gtattttagc tgctgtttctggggactgtt 4500 cctggtgcag ccacaggggc attctggggg ttgctccaga gggcagctgtgcctgcattc 4560 ccctaggtat catcgacttg ctcctgaccc actgccgggc caggtttcaggcctggcaga 4620 tgatggggaa ccctactggg gccagccctg cgttggcccc aagggctttctttcttgata 4680 ttggacatga ctgtggactt caccccttct gaggccttcc tgggtgccaagcagtgtgat 4740 ttctttcttt ttctctttgg tttgaaacag aatctcactc tgttgcccaggctggagtgt 4800 agtggtgcag tcgtggctca ctgcagcctc cacctcctgg gctcaagtgatcctcccacc 4860 tcagcctcct gagtaactga gactacaggt gtagtcccac ctggctaatttttgtatttt 4920 tttagagaca gggttttgtc atgttgtcca ggctgctctt gaactcctggctcaagtgat 4980 ccacctgcct tggcctccca aagtgccggg attacagggg tgagccactgtgactggcca 5040 gcagtgtgat ttctaacttg ggtcctgaac cattcaggcc ctttctgagtagctttctag 5100 tcaaccttcc agatgtcagc tctggctctg ttctgggggt gtatctggttgtcctggttg 5160 ttctgggctc tgtcccagtg tgagtgtaga gtgggttctg ggagctgcagttggtgaatc 5220 tgagtcttta tttggttgcc tgataggttg ttttgaggtc agtttgatcccagactgacg 5280 tggttcaaag ttactgtttg gcttttctag gatcctgccc tcatgctgttctgtgtgcct 5340 cagggtcttt gcaaatgttt agtcttctaa aatggcccct ggccactttgggtacacacc 5400 tctttccagg tgtggcttct gctccatgta gggcctgtct cagtctccaagcacgagacc 5460 ctgctgtggc tgggtgtggc agcagcgcct ggccagcttg gggggcctttcccattaccc 5520 agttccctgc cttggactct gcccctctgc ccagggtctc tgcacaatggtgcagccaaa 5580 aacctcactt gaatactgtg aggccctgcg ggcatctcta gacaaatgggcctttaagtt 5640 tggggcctgg aagctggttt gtctggccag acctgctggg cagcctgtatgctgggagca 5700 gcagctgctg gaaactcagc ttggggagcg gggagggaag catgcagagagggctgcggg 5760 ttctggtgca ggtaggtggg gcagtggagg ctgggtcgcc aagctggcgagggccatctt 5820 gggtcagttg ggatggcctt cagtcggtga gagtaccccc atattggcagaggtggttag 5880 tgcaacttga gggtaattcc caggtgcaga ggtgtgtcct tgatttcatgatgagggtca 5940 gtcttgggcc ggtgaggaca ttcccacgca ccaccctccc actgtggtttctttgtgtcc 6000 gttataggag gagccaaggt ggcagagtgg tgtggggtgg ggatcactgtgcatgtgggg 6060 gcttgaggtg agaagcctgc tgcaaggtgc agcttgcgtc gagtaggccaaacagctgag 6120 cttccgtgaa gtggtgatga ggtggtatgg gtgtgtctga cccccacccccacccaggat 6180 ggtacccacg ctggtgggga ctcaataatg gctctagaag gaaggggctgggctctgtcg 6240 ctgcatgtgt gccagcggag gaggcatcca ggggcacgcc ttcattttaaaaattataga 6300 gtagagaaag tcaaaaataa atattgcttc tgaagcttct ttgaagactggcatgtatgt 6360 tggggtgtgt tcttccaggc tgggctgttt tctttgaact cattcatgcgttcatccact 6420 cattcagtgt atgctgactg agcacctact atgtgccagc tgtcagccaggcgctgggga 6480 tacggaaatg agctgggcag ccacagtcca tgctcctatc aagcttatggtagtgactca 6540 gtaattgtaa aaatatataa atagtcatgt ttgtggtcac ggctccaagccaaggtccag 6600 gcaggtagtc tgggacattg tggaaggctt ccctgagaaa gtgccactggacttgagatc 6660 cgagggtaag ttggccagaa gagcatgggt agcctgtgcc aggcagtggcattcagcact 6720 ggcaaggctg ggagacagga agaaatttgg cacatttgag gatcagaagaggaagttccc 6780 gttctggcag gaggagagca tgggcaggta gggctgcaga ggtgggaggggccgactctg 6840 cttggctctg gggtcatgct gaggagctgg gccacagagt ctactgctgaggtcagggac 6900 ttggtctgtg tggctcacag ctatatggct ggtgcctgga aggacgcccagcacatagta 6960 ggtgcttagg gagtatcaaa atgaatgagc gagggagtga agggaaaagatgggcatttt 7020 taccagatgg ggatggttct acatgaacat ttccataacc tgcttttttttctgcataaa 7080 cagcaaattt ttaatgtctt tctgtgtcaa tacatatgct gttgtgatgaaattcttgat 7140 gttttctcat gctttccttt ctgtccagga aggagtggag gccctggtgcccggcccttg 7200 gtgctgagta tccagcaaga gtgaccgggg tgaagaagca aagactcggtgagtgtgccc 7260 cacagagtgg ccaggagcag gggtgcacag gggtcctatg gaccaggctgagcagcttgg 7320 atttgatgca gggtcagtgg agagctatag agggggtgtc agcagggggcatgacatgat 7380 ccagtgtgca ctggtgagag attgtggagg cctggaccag ggaggcagtgacagaagtgg 7440 agagaaggga tgtgagtgct atttagggag gtagagtaga caggacgtggtgaaggattg 7500 aatgtggggc atgaaggagg ggaggtgtca aggatggtgc ctgagtttctggctggagtg 7560 gatgatagag gtggatgggg aatgacatac agctcgtcag ctactgtaggtgtgaaatag 7620 gcttccgggt gatgtcctgg gacttacacc aggatctttc ttctgcttctccattcgcca 7680 cgtttctgta gaaaagactg ttcccatcga ctgtaggttg tcgtagaaagagcttggtgc 7740 ctgcaggatc tgggcatgtc ctctgcatct ctttgatgtc cccttgttctcctctctgtc 7800 tgttagaagc acagaatggg tgggctggag ttgcagggaa tgaggttggtaagttggttt 7860 agagtcagca tcatgggcct tggatgtcag gctaaagact ttagactttttcctgagggc 7920 aatacagcag cattgaaggt tcttaagcag agaagcaata tccatgtgttgatctgtcca 7980 ttgcccatcc gttcattctt tcacggaaca ctgtctgaat gcctgctgtgaaataaagtg 8040 gcaaacaaaa gtgcagattt gcatttttat gtgctgaaag gtggcactggtctgtgtgcg 8100 ggatgggttg ggagtgattg aaggcaggaa gcccaccaaa gaggcaggaagtgatgaggt 8160 cttactaaca cagtggcagt gaggatggag gcaggtcaga ttcaaacttattgatgggtt 8220 agatgtgtgg gatgaggaca agggagcatt taaggagggt ttgcgggcttggtgactgac 8280 aacatggtgg tgctgtcccc aagactggac accctggagg aggagtggaattggggcaga 8340 tgagttctgg ttgggccatc ctgagccaag agctcggtgg gtctgttcaccctctgtaag 8400 ctcctcgaag gcagagcctg tgactctctt gttcatcacc ctgcccactgaacatctgga 8460 cgggtggctg aactaacaag gctgtggaag agatgggtct ggcctgggtaggggaaggaa 8520 atgtgtatgt cctgggtgtc tcctggggtc cagaccctac gtttggtgagatgggcactg 8580 gggttatgta gagggtgcag caagaggggc tgtgtcacag gctggaaaggtcagatgggg 8640 agggtgggtg tagggacact atgtgtccct ttgcctctcc atcctcagatgatggtctga 8700 cctaactcga agtccagtct tgccagaata ggtacctgaa ggtggagccctctgtcctct 8760 cgggaggcca ctagctgatg gcatgtctgg tggggtgcag atggggtgtgctataggacc 8820 tgacttctgg aggctgggta gggctgatgt gggggtacag gggaagatactctgagatcc 8880 tgaggccagg cgccagcaaa tacaggagtt aagccaggtt ggagcttccttgggtacaag 8940 gcccagggtg cccacagggg attgggttct ggggcagggg ccaggtcaggctctaggctc 9000 aattggcaaa ggatcttgtt gtctgtgttg gggtttctca gcctcagcaccattgacatt 9060 ttggatcaga taacccttat tgtgggggca gggcttgtta ttgtagggtgtctagcagca 9120 tccctggttt cttcccccta gatgccagta gcatctcccc gctttccccagtctcgacaa 9180 tcaaaaatgt ctccagtcgt tgcgtaatgt cccatggggg caaaattaccctgaattgag 9240 aacctctggt ctacagcctc ttgctgcttt cactatactg tacaaacctggaaaattggg 9300 gagggtcagt ggggaagatg gatagtgatg ggccacttga gcagagttctgaggggtgag 9360 tgagtgctgg ggcaggagaa ggacattttt ttccaataga cagaacggcaggtacagagg 9420 cttggaggca caaagttggg aaacagtaat ccaaggggga ccccagccccagaggaaggg 9480 gcctggaggc ttaggggtta cagccgcagg aagatacctc tgctggtcccctttggtcat 9540 ttgcctccag agttagtctc ctgctgaccc tttgccgcct ccaagtctctgcgacactcc 9600 tcctaatctc cccctccact cctcagtgag atggagaaac tgagagctgcaacagggtgg 9660 gactcagtcc agctggtggg agccccgata ccagccgtgg gagggagggctgttcctggc 9720 tctgctcacg gattctggcc aactggctgg aggagggaag gcggcctcaccctcttcccc 9780 acaggcccct ccttccctgg gtcaggggtc ctggctgaga ctataatttattcccgtcat 9840 aatccagtgg ttggtttggt gagagctgga aacatgttgg ttttcccttcccaagtataa 9900 caaggcctgt ttccgcctcc gcggccgctg ctgcagtgcc acgcggtgaactgtccagga 9960 catgacaaaa gggctcggtt agctgcccgc tggttagaag atgaacggctcagagctctc 10020 cctgccgaca ggctcttccc ttcctgttgc ccaagtgctg gcctttcctcttggccgtct 10080 gctctgtagg gccctggata cccctccttc tgctcatgtc catttctgggccccagggtc 10140 ctggcctggg gaggtgagat gggggaggct acagaaacag gtggtatttggagactaatt 10200 aatatttgtg aataataatg catgcccaag gaaaacaaat caatctgtgtttgcgtattt 10260 atgatgagga ctcaggaatg atcccagctg ttctccagcc agggaagcccccatcacatg 10320 gtcccttggg cccccacaga ggctggcagg gtggttaggg tgggctgcatggaggtgaca 10380 tggcttctgt gttttgcaac gtgaatcttc tgtgacgttc atatgcgaatgctgtggctt 10440 ggtctagtgc ctgcttcttc cttttccctc aaagcagtga ttcccaggctctttggtttc 10500 acgaaccaat acaatttcca aaagtactca agagccagac atggggttggcaatttttta 10560 ttttgccaag taaaggcatt ataaaaaaac aactgctatc tgctgtccccatcatttcat 10620 acaggaaaga acattttaac accaaggaca atatttgaga ataaaggacagttcttccca 10680 agaaagggca gttggcagct ttacactgac gtagcaaagg aaaaggatatattaaaacca 10740 ttctgaatgt ggaaaaattg tgtccaatta tattctataa tatcataccgtatgtaattg 10800 tgttcctgcc caagtatgtc agcttggcag aacggatggt ccacagtgcaattcagagac 10860 gagcttgagt gccccagtgc tccggccttg tttttatctc ggttgttgaaagcccaggct 10920 cacccagcca tgctggtgaa gtgggtgttc tgctgtgaac accgttgcatggattcacaa 10980 gatgctcaac ctcgcaaccg cagtcaggct agcagcagcc ctgccccttgttcctctccc 11040 accttctccc cacaggtgac cctgggaccc tgggactctg ggggccaaggtctactcttg 11100 aggccaggcc tggggatcca gggctgctct gctctgatga gggaggccctggagctggga 11160 gtggcctgaa ccgcctgaca ccagggcagg ctctgtgccc ggagtctcagggcgggaggc 11220 agcttgctct cgccaggagc tggtgaggga gaggccctgg ctcgtacagctgtgtggctg 11280 ccagagtagt tgcctgagaa tgtgtttgtg tgtgtccctt gtgtttccccatctccctgg 11340 gcatctgtgt ccttgtgtcc atcatggacc actggaagaa gctctctgttctactttctg 11400 gatctgaaca aagtgtcctg agcaccccaa cccagataca cagggggtttctggaggccc 11460 cacgttgggg gtagggttac ctgaggccca agttctccaa cacccatggccagactctca 11520 tccaggcttg taccacatgg gtgtctattc tgggagtttt acccccatgatggctctcct 11580 gggagctctg cccccaccat gaagtctgta acagcacgtt acacctctgctaatgtatgg 11640 ggagttgcac ccctgtaatg tccttatcag gcagctgcac tccatgatgtctgtaccctc 11700 tgtgtccgcc cacgtgatgt ctagaccaca ccattacacc tccatgatgtctgcacctgg 11760 gcgttatact gctatgatta ctgcaccaag gcggctacag gggattaagcctctgctcat 11820 tgtactgggg agctatatcc catgatgacg gtgctgtgca ttttattttcatagcaggga 11880 gatatgctca tgctgggggc ctatgtccct atgaagcctg cctggcaggggttgcatgct 11940 tggaatgctt gttcctggaa gttaacctct gtgactatta tattgttatacccgatgatg 12000 ccggtacgag gccactgtgc cctgtccttt ggtgcaacac agctgttgagttataaactc 12060 atgtgtctca ttcaggggct ccaggatggt ccatcagggg accatacccccatgatgacc 12120 tggcctggca taggatacca gatgtgtcca caggcccagg gaggaagagccactcatagt 12180 ccaccacctg atggccgctg ggaaggtgct ccatcgttgg ctgcatgtggcaccatagcg 12240 caagctcagg cagggccttg gtgatgtgtg acctccatcc cactgtggccttgcaggcgt 12300 cattgtggtc tcagccacct cagctctggg atcgtggagc agatccatccccagcttggt 12360 gtaaatgggg ctggacacct atcaacgttc ttacttgaag aactggtgtaaatgaaggct 12420 ggacaccctt caccgttctt actttgttgt tcagctcctg tctcagatttttggtgggat 12480 cctgggacag gggacaactc tcaccccttt tcctttgtgt ttggccccatcccactttgc 12540 attcccacct gctttgtcca acacctttgc tcaactgctg ccttctgacctcatgaactt 12600 tgattatctc agttgagaca agggttcaaa tttcagaaac gactggagcacctttgagcc 12660 ctgcccattc ctggtcacct gccctgtcac cttgcaaatt ctcttactattgtgggtcag 12720 cggggacctg ggtctgatca caactcccct atcaaagtcc atgcgtatcactgtggggcc 12780 ccaaggcgtg ttcaccgtca tgcactctgc tctgttgcgt cttcacagcattcatgaggg 12840 atcgaccacg tctcacccct ctgcccagag catggtgtga tcagagcttgtgggcccttc 12900 catacatccc ctggtgcccc ccaggtgagg ggtatgggag agagaccctggggagctcac 12960 cagtggggcc agggagtgat agcagcgtat ctcccatgta tagtgcggccatgctagagg 13020 cttcacccag gtgcctgcag gcacttcagg ccaggagcct tggaagtaaaggcctagggg 13080 cattcagcca gtcccagacg ctactcagtc tttttgtgca tagcttcctgtctcaaacat 13140 cagcccctga ggttcacacc ccatcatccc tacacttgtt tggctccgggcttctggggc 13200 aagggcagaa gagatgtgag gtttgaattc tagggtctaa caccccattcctggccactg 13260 aggcctccct gtggtttctc tgtgaagtag gaaggtgcca tgggaatgggtaggacgagg 13320 gtggccaggc agggcagcag cagcttgtca agagcacgtc ctggtgagcactgaggggtt 13380 gtggctttgg ctggaagcct ctgatacctg gagcctgtct tctgctaacaagcctgtgtt 13440 caggggcctg tgctcagcgt gctcgtggag tctggcctcc caccttctccctccctgctg 13500 gggagaggag gggcaacaga attctaccag ggagactcca gagtcagttggctttgcccc 13560 tcactgctcc ttgtcactgt cttgtctctc tgtcctctgt cccacgtaagcgtctctctg 13620 accctgtttc cctgtcctct tatgggggtg agttagaagc tcaaggtttggactggaaca 13680 cactgtacat cctctggtct cactcactcc ctgtacctcc tactcaggtttagagaatgg 13740 cccgaaggcc tcctgcgctc tttctccctc caccgtgact gttgggctgtccttgtgggt 13800 gctggggaat aagtgaggag cttgggtggt cactggggtc agaaggtctaggtggtcatt 13860 gcacagccca agtggggggc tctgttgaac taggctctgg atggtcagtgaggatgactg 13920 ggggtcagaa agcgtcaggg gtgggcatta gggacagcag taaataccagctaactggct 13980 ctgcccttct ctccattaca ggttgattgt cctgggctgt ggctggctgtggagctagag 14040 ccctggatgg cccctgagcc agccccaggg aggacgatgg tgccccttgtgcctgcactg 14100 gtgatgcttg gtttggtggc aggcgcccat ggtgacagta agtctgacccctcaaggtac 14160 agatctccca ggttgaaagg tggcatcctt cccagcagga ctctgggatggggacagacc 14220 ggctactagg agagacaggg tggccagaaa ccctttagac cttctgtcctagagagcccc 14280 cactgcttgg agagcctcct tctaaggaat acctgggtac ttaggagcatctttaggcac 14340 cagagcgggg cagtcgaatc gtgaccctgt cttatggggc tggggtgagacctgtcctgg 14400 attcatgtgg tcattgtgtg atgtgcgcgt gtgtgtgctg tgtgcactgttgcagatctg 14460 tgctgaaggc acagtgtgag ctacagccag gggtcggcat gggaatggggggtggcacgc 14520 agggcatgcg tatggatgtg tgcccaggac gcgggtgtgt ccataggcccaaggagggtc 14580 aaaagcggga gccacccata gctcttcccc tacgctggac cctcaggatccaagtatgca 14640 gatctgagcc cctccttctc ctttcatggg gaccttctag gcaaaggagcatccccccac 14700 cccagctcct cagacctgga aatgggggga agattgtgtt tggccggcaggaagctgagc 14760 aacgcccctc tgtgtgtgtt tgtgttggag ggaggtggaa ttgtccccatccagatccaa 14820 ctccgaactt tggtcccttt cctgctgccc ttccccctgc ctggcactatgagactggcc 14880 tcagcccgcc ccatcgccat ggttaccact ccttggttac tggtgggaggcggggcacag 14940 ggcagattta gccaatctgc aggctgaggg ggaggggcga ggtcggagccaaggtccctg 15000 ggggaagggg ccgttcccag cctgtccaga gcccaggggt gatccagggccaaccctggg 15060 gtcagcccac gggtcaccag gccatagggc tcccccacct gcctccgtatctctgggtac 15120 agatctctcc ccttgcccca ccagggctct ttctcctaat ggctcattaattattcagga 15180 actgattctg gagtggggtg ggactggtgt ggtcccgccc tttgtcctcaagtgctgggt 15240 cctgggtgga gctagaaggg aaaggagaag gggcaggcat tcccaaggggtggggcaaag 15300 ggtcatggga gcaccaggta ccaaagagag ggctgggcag gtgagagcaaggaaaaaaga 15360 caggtgggga atcacatggg gaagtggggc aggggatctc caggcctggctggaacctgc 15420 aggggcaggt tggagtagaa agccttatca ggtgtgaccc acatggttggcaggaaggtg 15480 aaggcttgtc cacgtgtggt cagtagctct tggacattga agtactgtccttgccctccc 15540 cgccgaccca accccaggca ctttcagggc cttcagacag gagctctagggcaggggaag 15600 gatggcccag ttgggcttca acagataaca catcctggga gaagagctgccctcctcctc 15660 ctgcctcaga cccaacgcgc cccaacctgt acccctcact ctcatcccccagcctgctgt 15720 cttcccgtgt tctctcccgt gaaaggcatt accacccacc cggtcacccaagtcagaacc 15780 tgggcctcct ccagcctcct ccctccgtga tatcccacat ctaatccatcaccaagctcc 15840 gttgcttcta ccttctgaat atctttggaa tgcatccact tctctctctaccgccttcat 15900 ccaagccacc ctcggctctt gggcttttgc acgcaacagc ctcctgacggtttccctgcc 15960 tccagtcttt tccttcctaa tccattctgc attccaaagg tagagtcgtgtcacaaggtg 16020 aaaacatcat cctgtcactc ttcagtttag aatccttcat tccctcccacccccaggccc 16080 ccagggtcgt aacccttcta taggccctgc attatcaagc ttctatcaacctcatccctc 16140 gctgtccctc cacccacccc actcacccac cacgctgact tctttcagtggttcaccaat 16200 tcccagtagt aattggtagg ggcaaggggg aacagttttt aaagcagagaacctccatgc 16260 tggttcctta ggaaccaaag cccaaacacc aggtcttgac ggtagaaagcagctgaagaa 16320 gctgaaatcc tgccttcttc aggtaggggg ttcaggggac tcaggccttctcccaggcag 16380 agagccatgg ggcaggagcc tgggctgggg gtcagagtga ccgagccgcaggagccaggg 16440 tctgtcttcc aggcctgtct tacactgcac acagttttgg atctctccctgcactgaggc 16500 agggctcagg ctgagcttgg gcaactacta gccaggggca agtggccatacacagacagg 16560 gccactgcag aagatagggt aggcacccca gagaattgac agctggggcagtgaagcgcg 16620 gagtggacaa atgtgtttca ataggcctct taacgagaca aatgaatgggggccctggcc 16680 tctgagggga gtggagggaa gcgggtggag aagcagctgc caagagttagcccagaggcc 16740 ccagtgtcag tgccgcacgc gcagctccag tggagatttg ggcacacattggggtaggat 16800 ctgctgcagc gcagcttccc cagccaggct ttgtggcttc tcaggaggggaggttgtggg 16860 gccagaggtg tcctgagcca gggcagaggt ttttgctgat ctcaagtgcgtgtcgcgtgc 16920 ctgtcttcag gcaagacctg cagtgtggag gcacaggctt gtgagcagtgaccacagggt 16980 gtgctgggta ggtggcactg acacaggcca tggcagaagc atcttgggagaggagttggg 17040 aggcttcctg gaagagggga gccctgaagg gtgagtggac atttgctaatgggggggtat 17100 tgcataatga ggtggggttg cggggaagaa gcacgaatgt ggctgggcttctctgtggaa 17160 ttcatgggag agccacagtg agaacccgac agcatgggac agaggtggggtccgaagacc 17220 aggtgggagt gagaccctgg ttatggccca tttacccaga atcctaggagctcagagcag 17280 gatgcgtgct ggctggaggg gtgggtggcg ggtgggttga cagaggtgggcagaggctga 17340 ggagctggga gtgtgtctgt tgtgtcattt ccctcctccc cagagcctgtggagcacaca 17400 gggtctgttg tctgtcgtca tgctctcccc ctcgttctat gggtggcctgtctagactct 17460 gctccctgtg ggacttcccg cagattccgt tgctttctct ctctgggcctgttttcctat 17520 tgcacaatgg ggataatcac tcctacctgg aaaggttaac tgaggtcacatggatgaggt 17580 gcctggaacc tagtatgtat tccccttatc tgaggccatg acctggggctcttgctctgt 17640 ccctgggaac caggcttgtc tctgagtggg ctccaggggg gtaccaggaacagtcacagg 17700 agctcactga gtcccagctt aagctgctca gacccaggga tatctgtctctccagaagct 17760 ccctgccctg ccttcgccgg ccctcatggc cctgcctccg tgtgtacatgtgtatgtgta 17820 ttccatggga aaggcacaaa atagcagtca gtctctccat agaagagccttgatggtggc 17880 ccagtttgac tctccctggg gctggacccc tacagcctcc ctgggaggtggttgcagccc 17940 ccttcctcca gccagttcca cttactcctt tcttaggcca cttcctcccaccctgcatgg 18000 gcttggtggc tcgagaatgt tgccgtccat accccgggag ctgtgctgaaagggctgtgc 18060 ggcccccgac cactgtgtgt gtcagggagg gggcacgctc tcgtggggtgtcaggccagg 18120 tggcagtggg taactggcag aaaggccctc ctggtgtgct ctggtggcaccctgttgacc 18180 cagtctcaga agttgtgttc cgaccctcac tgaacaccag ctgtgggtcaggcacggggc 18240 agagtagttc aagtagcttg gtttgctgcc tgcctgggga cctgacactgtgggatctgg 18300 tcagtgctgg gatgggaagc tctgggcacc tcaggccatg ggacacagagcaggctccta 18360 cagcagcttg gctgggtggg acatgagaga ggggctgggc tgggcacactcaaaggcagg 18420 gaggagtctg agggcctggc ctgtcagggt ggcctaggtg gtgggtccaagctgtgtgct 18480 ctgcacagtg ctaggcctgt actatagtag gtgctcaaaa aatacttgttgaaagagtaa 18540 agaagccggg tgtggtggct catacccgta atcccaacac tttgggaggccaaggcaggt 18600 ggatcgccag agctcaggag tttgagacca gcctggcaat gtggtgaaaccctgtcttta 18660 ccaaaaatac aaaaaattag ccaggcatgg tggtgtgcac ctgtggtcccagctactcgg 18720 gaggctgagg tgggaggatt gcttgagcct aggaggtgga ggcggaggttacagtgagct 18780 gagattgtgc cacttgtact ccaacctggg tgacaaagtg agacccccctctcaaaaaaa 18840 aaaaagacta aagaaaagtg agcctgagag cttaggagga gcacatttcagaggggaacg 18900 gagagaggaa catcaggccc gttggtagct gaggagaggt gcggttagatctgtgctccc 18960 caaagatcct ctgctgaaca taaggggcaa cgccttgtct cctgtgctgtgtcctgcggg 19020 tggaggtgga ttggagggaa gcggagggcg aggcctggtt gaggggcggggcctgcctgt 19080 ctggtccccc gggctgcctt gggccagctt ggcctagtct gttgggtgggcgggcagggt 19140 gcaggctcct ctccagcctc caagggaggg gagttgttct gcctcctcgatagccccagg 19200 ccttgggcac agcccagcct cccacggctc ttgggccctc ctccttccaggccgccggtg 19260 acccacacct ggctctcctc cccggcgtct cctctccgct tctttgtttggagcggaggc 19320 cccgccccac cccgccccca ggcgcactcg cccggccatt ccggttcagccggttccagc 19380 ccccagtttc tgccgctgca ggtcccggca ggagctggag gggcactttctccctgggtt 19440 tctcttccct ggtgcagcag gggccgcggt cctcatcctc ctggttcctcagttcggtcc 19500 ttctttcatt ctccacccct gggtgccagg aactgggtca gacactgggacaggaatcca 19560 gacaggcatg ctatctgccc tgcccagggt tatgttctag gaggggaagcagccattaat 19620 caaacaccaa aaatgtggaa aagtaataat ctcacacgtg tgcataataaactgtgagtg 19680 aaagttataa gctcggcagg taggtaataa gctaggagca gtgctgtgggaggcaaggga 19740 gttacccggg agtttcaaac taggaactga gctcataggt tgggggcagggggactggag 19800 aaggcagtga tacttaaatg gagagcagaa ggatgaatgg aagttagagtgtatggcgga 19860 ggttggcaga agcagcagct tatgcaaagg ccctgtggct gcagggaacatgacgttgct 19920 ctttagagga gccaaagctg gggctctggg gagagcagct gggtcagaccccgcggcttt 19980 gtctgccata acaggtgttt ggagagtgat tcggcaggtc tttggagggttttgataggg 20040 cggggtgtcg ggggaggctg tcagctcact ctggacactg agtagagaacagacgggagg 20100 cgtggggcag gcctggaggc aggggcttcc gcgtgttagg ccagtggagtgccagtcaag 20160 ggaaggtggt atctggacta gggtgtggca gcgcaggtgg agaaccctgagctgctttgt 20220 ggagggcttc aagtgtgggg gaagagtgca cggtatgggg gtgggggacaggagacaccc 20280 ccagtggagc ctgagcagga gagtcgtgtc tgagagggtc tgtctggaaggcgcaacaga 20340 ggccaacttt gcagacagct gcagtcggga gagcctggag cctccttcaaagggcattca 20400 ggggaagggc aaggcacgct ggggggttct ggaccttctg tggtgtcttcttgtctttct 20460 ggtccctaca gcctccctga gctggctgcc cgagcctgcc ctaggcactctaagaacata 20520 gtcagtccca aggtctccct ccagggaagg ccgtaggtga gcttaggagtgagaaggctg 20580 gatcaaagcc tggctccatg cctgcatccc tctgacttgc cagtcatttcaccctccgag 20640 cctctatttc cccacctctt aaatggggat aataatacta cctaccttatgggattgcgg 20700 taagactgat aatgctggta cgagtgacag cttccctcat ggaaggcccaccacgtaaag 20760 cagtttacag ccatctcatt ccatctatga cagaatcctg tgtcactgttttggagatgg 20820 gaaaaaagag gctcagagat ggtgagtgac ttgcataata attacataaaacccccagag 20880 cccctggccc ctggagttct caaaagttcc ttctctgttg gtacctgcagctgccactcc 20940 ctaccccgct cccatagacc ctctcctcct tggagactct gccccatctgccgtcccttc 21000 tctgctggat caacaccttt tccctctctg ctggctccca tcagtatttaaacattgcct 21060 ttcatatctg tcttcaagaa aaaaagaaaa aaaaaattca caaacctcccttcccgcctc 21120 atcctttcca gctgctggct gtatagtcac ctgtacttcc ctctctccctcatcgcctcc 21180 cagtcattct ttggccttct ttggtctggc tttggccccc acccaccactccactgactc 21240 tgttcttgtc aaggtccctg acaatcttgt gtgaactgtt ttgtaccaggtgtttgacag 21300 tcaaacatgc ctgaattcag gtcccagatg tgcccctcac tggcatgtgatcttggacaa 21360 gtgacttgac ccctctgagc ctgtaaactg aggataatag caatgaaggactaaagataa 21420 agaacctggt gcagagtggg tgcttggcaa aggattgtca tcatcgcacacgtttctgtg 21480 ccagggacca ggctgggcct gggctcctgg ggaccaaaca ggtggtctgaaaggtcattt 21540 ctcacagcac tagccctttt tggagctgtt cattggtctg attaataggaaatggatcag 21600 ctgtcaagat taacgagcta ttgctacaag attgtagcaa atgggttgggcttctctggg 21660 ttcatgaccc taggtggttg aattcttagg gataggggct gtggactggccctggtatgt 21720 gtactgaggt gatgagggtg tggcagtgcc atgtctgagc ccctacctttcttctcctcc 21780 ctctgcctcc ctgtggacac cttgaggaga ctgtcagaag gcaataactaagtcgggggg 21840 gagggatggg agaggcagat ttacaggaaa gcattcacct gggaagatatccagagagac 21900 ttaggaactg gactgtctag gcctttggga ctgctgctgg tatgtgggggctgggagaga 21960 gggaggagtc tcggttcctg gccggagccc cggggtggat ggtggtgccatcactgagat 22020 ggagagcagg gggagggaca actctcaggg agagctggag ctcttcccagcagctctcca 22080 gcacgccttt ttcctggagc ttggaattga tgtggggcgg gcaacagaagggtgcagtgg 22140 tagtgtgaac tccagacttg gaacacctgg gttcagatct tagctctaccacttaccagc 22200 tgtgtcatat gggacaaatc ccttaacctc tctgggcctc tagaaacagatacagttata 22260 gcactcacct catatgctta acagagttaa aaaatgttaa actctctgaacagtgcctgg 22320 cacatactaa gcgctacata aaggtgaggt gtccttgttt tctttgtaggtctttctctc 22380 tgcccccatg actgccacct tcctcactgg ccattcctat agtgactgtgctgtggtgac 22440 ttggtgtctc catctcttcc aggcaaacct gtcttcatta aagtccctgaggaccagact 22500 gggctgtcag gaggggtagc ctccttcgtg tgccaagcta caggagaacccaagccgcgc 22560 atcacatgga tgaagaaggg gaagaaagtc agctcccagc gcttcgaggtgcgtctgtgg 22620 tgggaagggg tcggcagggc tcagggtctg cccacactct ctcctttcagtgtccctcct 22680 catggacctt ttggaggtgg gaggacaact gaccctgagc aggctcctgtgtcctgagta 22740 ggctgtgacc ccatgtctgt cctctgacag gtcattgagt ttgatgatggggcagggtca 22800 gtgcttcgga tccagccatt gcgggtgcag cgagatgaag ccatctatgagtgtacagct 22860 actaacagcc tgggtgagat caacactagt gccaagctct cagtgctcgaaggtacgtgc 22920 tagggagacg tggcacggtg ggctgccggg ctgaggcgtg ggaagagccagccagccctg 22980 atcctgtcct gggcccatgt gcatttggca gaaaggagga ctggccacctcggggtcagt 23040 gaaagtcagt ggtggacagg gatagtcatt ggatctggcc tggattgtgcggcttatgct 23100 gaggccagcc atgtggggca tgatgccttt gtattctcct gctgagccgggtcgttggtt 23160 gggtggggtc tggggtctga cttgaggtgt ggagctgcag ctgtgtatcccttgggttac 23220 gtggttatgg ctgtggctgt ttggcagtga accggatttc catgtggagcctggccgtag 23280 gtgtcaggca ggtgtgttcc ttgttgcccc tgtgagctga gggctggggctctgtccgtg 23340 gattttagtg tcttctctca cttggtggct tctccattca ttcacaaacactccctggac 23400 caccttgaag tcctctgagc accgaggagg aggaagctgt gtctaagccaagtcttgagg 23460 acaggtggga gttgggggtg gcagttggca gctaggcagg tgcccaggcccagaagcaag 23520 agaggatgga gctttcagag agctctgagt agttcaattt gggttttctggagggcagag 23580 ggggagctag agagcacagg aagaaggaga aagcaattca gcatgagtctggagaggttt 23640 ggagggcaga ttacacagga tctggctgag aaatgaacac tctcctagggacataggaag 23700 ccacaaacaa ggcgggggtg acatgatcag accccagcca caactgattcatcagtctga 23760 atgtgtttat gttttcaaaa tatagcatcg attgttgctt gcttttttttcttttctttc 23820 tttttttttt tttttttttt tgagatgcag tctcactctt gttgcccaggctggagtgca 23880 atggtgtgat ctcagctcac tgcaacctct gcctcccggg ttcaagcgattctcctgccc 23940 ggcctcccaa gtagctggga ttacaggcat gcgccaccat gcctggctaattttgtatta 24000 ttagtagaga tggagtttca ccatgttggt caggctggtc tcaaactcctgacctcaggt 24060 gatccgcctg cctcagcctc ccaaactgct gggattacag gtgtgagccaccgcacccgg 24120 gccgattgtt gctttctttt taagaatgtg atgtcgatac ctgttcctttttgaaaaatt 24180 ggaaagtata gaatagcaca gaggaaaaaa ttaaaatgtc tcagtttacctctagtaata 24240 acatttggtt acttactcgt ggcccatttt ctgtgcatac atatatatgtgtgtgtgtga 24300 acagaaatgg gaccacatac tgtccgatga tttgtagact gctttaaaaacaaacaaaaa 24360 aaaatatggc taacatcttc cttgccacta aatattcttc tgtatcattattcttttttt 24420 tttttttttt ttttgagacg gagtctagct ctgtcaccca gcctggagtcccgtggtgcc 24480 atcttggctc actgcagcct ctgcctcctg ggttcaagcg attctcctggctcagcctcc 24540 cgagtagctg ggactacagg tgcgcaccac cactcgtgac taatttttgtatttttagta 24600 aagacggggt ttcaccatat tgaacaggct ggtctggaac tcctgacctcgtgatccgcc 24660 caccttggcc tcccaaagtg ctgggattac aggcgtgagc caccacgtccagcctgtatc 24720 attatcctta atggctatgg gtaagctgtc acatgcaagt accctaatttatttagccat 24780 tcccttattg ttggacacat atgttctcag ttttttcgtt tctataaataaggtgccata 24840 cacgtcgttg cagatggatg tgttcacctt cctgattatt cccttattctaggttcatgg 24900 aagtggaatt gctgagtcaa agggcacatg catttttaag ccttttgatatttccagaag 24960 attgtgtcaa ttcatactcc tgccaagcag ggcagaagag ggcctctttcctgcacatct 25020 tccccactgt tgggaaatat cttcaaaaaa atattttttg ccaagttaataggcaaaaaa 25080 tggcatctca atttaatttg catttctttg attacaagaa cagctgcacatgttttcaca 25140 ttggccattt ttacgctgtg gctttatctg ttcacacaca catctccattcagttactct 25200 ttttgttgtt gttgttgttg tttggttttg gggggttttt tagttttgtttgagacagag 25260 tctcactctg ttgccaggct ggagtgcagt ggtgagatct cggctcactgcaacctccac 25320 ctcccgagtt caagtgattg tcctgcctca gcctcccaag tagctgggactactggcagg 25380 tgccaccacg cccagctaat gtttgtattt ttagtagaga cggggtttcaccatgttggc 25440 caggctggtc tcgaactcct gacctcgccc gccttggcct cccaaagtgctgggattaca 25500 ggcctgaggc actgcgtcca gtgtcggtta ctgttttgca tctgtgtcttgtatgagtcc 25560 gtgggctcta caagcttaga ggagccatag aggacagtgg ttaagactctggaccactca 25620 cttctcgtgt gtctttgggt tagtaactat gcctctgacc ctcatttgctccatctgtaa 25680 atggatataa tgcctaccac ccaagctggt ttggaagatt gaatgctgtgtatgaagcgc 25740 ttcccaggag tgtggtggtg gttgctgtgg ggctggaagc tgtacatcttgaggcctcac 25800 cacgtgagct gaaggtggtt ggcctccgtg ctgcgtggca tcatccgggtgatgcagctc 25860 cactgcctgc tcccacgggg ggacatacct gtgcaatagg aactcagagaacaggccctg 25920 ggccagcatc actgattgct gagtgttctc aggctcctcc ctgccacttctgacctgttt 25980 ccccactcgt gacctggggg caagtatttc aatgctgccc agtcctcctattggcagctg 26040 tcctagcagg gagccatctg ccctgccctg gcatttggga ggtggctgaaggaccagagg 26100 ccccagaggg cttcttttca ggctctcaga ttggggaaca gggcctccttgttagtattg 26160 aaagaggagt ccttcaaaag cgcctaagcc ccaggtcctg ggtcaggagccctcttacct 26220 ctgccactgc cctgggcagc cttacctttc aaagggttga ggaggatggaggtggtcttt 26280 caggcctggg gtgaggagga cggacttcag actgacagga ggggctgggtgtggtggctc 26340 acgcctgtaa tcccagcact ttgggaggca gaggcctgca catcacttgaggtcaggagt 26400 tcgagaccag cctggccaac atggcaaaac cccatctcta ccaaaaaatacaaaattaac 26460 tgggcgtggt ggcgcacgcc tgtagtccca gctacttggg aggctgaggcaggagaattg 26520 cttgaaccca ggaggaggag gttgcagtga gctgagattg cgccactgcactccagcctg 26580 ggcgacagga gactccatct caaaaaaaaa aaaaaaaaag aggacctgcaggaggtgcct 26640 ccgtttggtt tctctaccag gggccagcag tatatgtttg cgcttctggggtgtgagaag 26700 tgtctgatga ggctggggag agtgtggcgc aagtacccag accctgtccatgctccgggg 26760 aagggtgtgg agctgtgggc ctgagtgagc ccctcctgct tctgaaacatagcaccagga 26820 agacaaaatg ctgacttatc ctgctggtct gtataacctt ggccagcattgggcttgaca 26880 gaaaggcgtg ccgcaggagg gccagaggga agcagcccag agggtgaacacccgccccct 26940 gccccgcctc accccacccc tcccctccct cctaggagga tcaagaaagctgggtgcctg 27000 caggaactgg gtgaaaggat ggttggactt ggactggagc tctggggtggggccggcagg 27060 agccagggcc attcttggga ggccaccaca gggtacgagg agggctggggaggggatgga 27120 gctagttggg gtcggggaag taagagtcct tcctgagttg cacacagctctccctgtgag 27180 ctggtctcct tggagaatgg ctgagggctg tctggggtga actggctcaggtgaagatca 27240 gtgtgccccc aaaaaggagt ccaggcctgc agtctgttct gtgccctctgcctttgcctc 27300 atgtcacacc aggcccaact gctgtggctc cagggccact cccaccacagcccgtgggag 27360 tgccccccca aatcccccac agccgtgccc tttgcacctc gcatctgagcaggattgtta 27420 ttcccagtgt ggcctcccct tgacaccccc gccaggactg catacgaggtgggggtgccc 27480 agcacaagct ggccggggtg agcctgtcct ggctgtgatg agcgtggggcccgccgccca 27540 gcgttcctgt ctgagtggta attgaagcca ttagcgcgcc agcctctccctcgccgggta 27600 atggcaggaa aagctcttct cgctccgcac tcttgaggcg gcggctgaatcactccccct 27660 ccaacccgcc cgctgccgcc actgagacag ggaatctgac attttccctcaccagggagg 27720 gggagccctg ggggagggga gggaggcagg cctggattcc tggcctttccctccaggagg 27780 ttgaggggca gtgaaggtct tggagctcag tctgtaagtc atggattcacctggggccgc 27840 agatttcagg cctggaagga ctggccggga aagcccagga ggccgccagacattcagtgg 27900 tgtggggagg gcactgatga ctttgggtag gttctgggag ggacaaggaggcggggagaa 27960 gaaagaagct gtggagcaga gatgagctgt tttggacttc tcctggggggcttagctcca 28020 agggttctga gtacaggtag cattacttgt cttggggtca ttgatagggcagataacgtg 28080 cactgttcgt gtggtgtggg tgccctgtgt gccatgttta aggcagtgtttgtgtgaagt 28140 gtgtggtgca gtgtgtgcag caggggtgct gccttgcaag ctgtgcgcacagaacggggt 28200 gtgcagcggt cagcgcacac gttaccatca ggcagtgggg aaggggcagtgtgtgcagga 28260 ggcactgcat gctgtatggg ctgtgtatgc agcgcgcagt gtttgtgtgcacgtgtgtgc 28320 gtcaggcagt gcgtggtgtg gtggtaagca ggtagtgagt accttatgggctatgtatac 28380 agcaaggcat atgcatcagg cagtgtgtgg gcagtaggca gtgtgtgtgtgtgttatcag 28440 gcagtatatg tgtgcggtgt gccgtgcggc aggctacctg tgtttatatcgggtagtgtg 28500 tgtgtgttta tgtgcagcaa gggtgtgcag taggtgtgca gtagactgtgtgcagcaggc 28560 gtgtgtgtgt gtacagcagg tggtgtgcag caggcagtgt gtgtatgtatcaggcggggt 28620 gtggtgtgtg tttatgtgca gcatgcggcg agtactttgt gtggtgtacagcaggtggtg 28680 tgcggcaggc tgtgtgtatg tatcaggctc tgtgtgtgtg tgtgtgtgtttgtacagcag 28740 gtggtgtgca gcaggcagtg tgtttatcag gcagtgtgtg gtgtgtgtgtgcatctttat 28800 gtgcagcgag tagtttgtgg ggtgtgcagc aggtggtgtg tagcaggctatgtatgtatc 28860 aggctgtgtg tgtgtgtgcg cgcgtgtgta cagcaggtgg tgtgtagcaagctgtgtgta 28920 tgtgtcaggc agtgtgttta tgtgtgtgtg tccagcaggt ggtgtgtagcaggctgtgta 28980 tgtatcaggc agtgtgtgtg tgcgcagcgg gtagtttgca ggggtgtacagcaagcagtg 29040 tgcatggtat gtacagcaga tggtgtgcag caggcagtgt gtgtgtgtgtgtgtgtacaa 29100 taggtggcat gcagtgggca gtgcatttgt gtgcatgcag caggctgtggtgtgtacagc 29160 aggtggtgtg cagcagactg tgtgtttgta tgcatcaggc cgtgtgtgtgtgcagcaggc 29220 agtgtgtgtg tgtgtacatg cgcacacgca acaggcacta tatttgtgcagcaggccatg 29280 tgtgtggtgc atacagcagg tggtgtgcag caggctgtgt gcttgtacatgcagcacata 29340 gtgtatgtgt gttgatgtca gacaatgcat gtgcatgtgt gtggagtgcacagcaggtga 29400 tgtgcagcag gctgtgtgtg taggtgtgcc tcgggagtgt gtgggggtgggagtggacag 29460 caggtggtgt gtgccttaca acctgtgtgg gcagcagcag gagtgggcagcaagcagccc 29520 gagctgaagt gtggtggggt cagtctggca ctggcaggag gtcggagatgctgtgtctgg 29580 gctcattgtc tctgggaccc cgcaggatcc ctgtttggag ctgttggtccgccaggaagt 29640 tcgacagagt tggatgtggc cagaatggtc tggctgagct gttctcagcacctgttgtgg 29700 tcgacctcag cagctcctct gtagccaggc caggaggtgg ggtgcaccagatagggatcc 29760 tgcccagacc tgcctgcagg aggtcttggg accctttcct ctccctcccctaccccacca 29820 agcccacagt cccttctgtc tgttcccacc tggcctcagc gtccttccagcaccatacca 29880 cctatgccat gcgggccagg gaatccaggt gtctcgatgg agtgcctggtgttgccagcc 29940 ttggagcagg ctctggggaa gggctgtccg gcacatgaag tagtgaccagggtgggggct 30000 aatcagtgct gtactgggct tggacctctg ggttctgcag gagctctgaccctggctggc 30060 tttggggctc gtgggatgac gaaggacctc ttgatatacc ccctgaccactccacaacac 30120 agtccttttg gaaatagccc tagagacaga gggtcaatga tagacatatgaccctgtcct 30180 gaggtatcct gggctgggct ggaactggag cagagctggt gagggagcttacatttgcaa 30240 gatgggtaca tgtgtgtttt ctcaaggagt catctctccc tgctccacctccttccctgc 30300 tccaaacctg tatattcctt ccacccatgg tggaatgact tggcccaggcctgactttga 30360 tggctgggga gttgggtaaa gggctattgc aggcacaaga tggcttcccaagtggcaaga 30420 tgtgatccct ttctattgtc tacatgtgtg tctggggtaa cagtctgctttggcagaagc 30480 tggggctgtc aactacctga tttgcagcac agaggatgta gaagtggccccagaacctgg 30540 aacccaagcc agcttctacc tctcccttag caactgaggc atgacctgtatacccctggt 30600 ccagctgctg tagtcctctg tgacctgtcc ttacaccccc atgactgcagtagatttcag 30660 atccacgcag acatgaggtt ctacaatcat ttcctggagg ttctaggcctcagggatggg 30720 gatgggagag gtattaagta agttctcctg tttttattag ctgtgatctgccttgaggcg 30780 cccaagtgga ttcccttccg aatgggtgct cctgtttagt aggtggtactcctgcctctt 30840 ctgttagcca tggacactgg gcacttcccc tgtgcccagc ctgcccccctgcccctatac 30900 tcacaggtac ttgtaaggcc cactgtacgt gttccctagc ctcatgaccacggtggagcc 30960 aatggctctg ggaacaggct caccaggatg gcggtttaaa tgcccaaatgccctgtgcat 31020 gcacattctc catccttggt aaggtgctcc ctcctactgg tttgcttgctggggagagag 31080 gacttgtttt tttttttgag atggtgtctg actctgtggc ccagactggagtgcggtggc 31140 acaatctcag ctcactgcaa cttccgcctc ccgggttcag atgattctcctgcctcagcc 31200 ttccgagcag ctgggactac aggcacgcgc caccacacct gactaattttttgtattttt 31260 agtagagatg gggtttcacc gtgttagcca ggatggtctt gacctcctgacctcatgatc 31320 cacccgcctc agcctcccaa agtgctggga ttataggcgt gagccaccgcgcctggccct 31380 gagaggactt ttttgaaacg gagtctcact ctgccaccca ggctggagtgcagtggcgtg 31440 atctcggctc actgcaacct ctgcctcctg ggttgaagca attctcatgtctcagcctcc 31500 cgaatagctt gggattacag gcatggacca ccacgcccgg ctaattttttgtatttttag 31560 tagagatggg gtttcacgaa acatgttggc caggctggtc ttgaactcctgacctcaagt 31620 gatttgcccg ttttggcctc ccagagtgct gggattacag gcatgagccactgctcctgg 31680 ccaaatttta atcagagcaa tgacgtgatc aaacttaagc ttcagaaaggtccgtgtggg 31740 tgcatagtgg gggctggaag gaaaccaatg acaggttgtt gcagtccaggtgagtgatgg 31800 cattggcgca aatctgggta tgggtagggg aagatagggg agcagatcactagaaggtta 31860 gaaagagccc gggcacggtg gatcatgcct ataatcccag cgctttgggaggccaaggtg 31920 ggcagatcac ttgaggtcgg gagttcgaga ccagcctggc caacatagtgaaaccccgtc 31980 tctactaaaa ctgtaaaaat tagctgggtg tggtggcagg agcctgtaatcccagctact 32040 ctggaggctg aggcataaga attgcttgaa cccgggaggc agaggttgcagtgatatgag 32100 atggcgccac tgcacttcag cctaggcgac caagcgagac tccatctcaaaaaaaaaaaa 32160 aaaaataaga ttagaaagag tagaggcctg cctggctacc tgtaggattggggaggtgca 32220 aggctccagg ttcctggctc tggggctggg taggtggggg tgccatttctaagattcatg 32280 tactagagga ggaccaggtt tgaggaggcg agttggtgag tccagttagaaatctgttga 32340 gccaggtgtg cgcaggcatc cagggaggtg tcaaagaggc ctcgtacacatttgcctggc 32400 actccggggg acatctgaag ttggaatctt acaggctgag aaatagcaaatctcaggagg 32460 ccagagccag gacagaagga gccccaggaa ccgactctcc atgttgggaggaggttcctg 32520 agagactgga gggaaactgg gagtgtgtga ggtcaagaaa tgccaagagaaaaaagtgtg 32580 cgaaggagga ggagtggtca gcagggcggg gtgctgccga gaggcccggcatgtgtcctt 32640 aacagttagt tccgtggagc cattgttggc tttggtgttg ggagacagcagggctgagaa 32700 gtgagaggga ggctatcagt gtctgtagtg actggaaggg cccgaacctagggagtgaat 32760 ggatgacaag agtctccctg gagtaggact gaggttgggg agccactgaaggagagtcca 32820 gtgtgggccc gagtctggaa ctccagtgtg ggtcgggtgt atgcgcagggcatggcccct 32880 gggttcttgt ggtggggagg cctcaactgg gttgagtagg gcgatgtggggtgccaggga 32940 aacccgagga aggaggggat gactgggagg gggacgccgt gcctgccaacaggccccaaa 33000 cagctcagat tgaaaaacaa aacaggcttt taagatgcca agtttgatgaaatctgagtg 33060 ctggaagagg tgggagtttg ctcttggaaa aacagctgaa aatcgagactcaaagctgcg 33120 aagggagatg ggcccaaggc tccccaggcc ccccttactg cctggaagcctgggggtggg 33180 gctgggctct ctgggagagt gtgggagcgt gcagatctgg cagccttcgacttttggaag 33240 gcgtctggct ctggccctgc tgaggatgga gctgctggag gtggccttggcttctgtgga 33300 gcggctgaag ggcaggggga gccagcagcc ctgccaccta cgaggttccttcatgtgtct 33360 cgtccctgca tgtgtctcca ggcgcacgtg ttttggcata ggtgtgtctggctgtgcatg 33420 tctccacatg tgtgtttgcg tgtctctgtg tgcaccgcct ctggatctgcttgcatgcgc 33480 ctgtttccac aggtgctagt cactgcaggt gcccctccac atgcacatgtaggtttctat 33540 ttttacacct gtctgtttct ccatgtatat ttctgtgggt tcctggctgtgcatgtttct 33600 atggtgtctg tgtgttgaga ggcagggtga ggccaggccc aacactcaagcttaggggag 33660 gcggtcaggc tcacagacgg tgccacccca gggggcctgt gtctgtgtgtgcgggtgcgt 33720 gcctgcgttt gcacggagac gccacgaagc tgggtaaaca tgggtgaaaaggtcatactg 33780 acagacggtg ctgcctgccc caggaccctg caaggcgtct ccatctgtcagcttccctgg 33840 tgcagccccc tccctctggc ccagagactc accctcaggc gtccaaggctgagctggaca 33900 aagagacctg tgtgtacttt gtagggggcc tcagcagcct ccacccccatcttaggctcc 33960 tctgtcagga ccccaacaca tgccccagct cccaccagac tcgccttggtactgtcatcc 34020 caccaccttc ccccacaaca gcctttacaa aggcagtttt cccctcctccctggaaagct 34080 ttctgcctcc catgctcatg tgtttcctgt tcttgaatct ccctcctccaggaagccacc 34140 aagatagcaa gtgagctgtg gagtcaaacc gatcagctcc aacactgctgtgggaggtta 34200 gccaagcgct catccacttc tgaaccttgg attcccacca tcgacccccgaccctcccct 34260 cgctagggct tgtcatcgtc ttctgcccat ggggcaacca aacctctccacggaagggga 34320 caggtctcct tgctgcagtg ggtaaaggcc agcgcagtta ggtgcaggaggcattcacac 34380 acacgtgcac actccccacc ttgcacacat atctgcgtga gccggggagaccctagggaa 34440 tgtgtgtgca tgttgtctat gcatgcgggt agaatccgca aacggtgtggagactcgggc 34500 tcttgggtac ctctgaaggc ccctgaagtc cccatgggct tctccttccgtccagggcac 34560 cctcttatca ggccatggcc ctgagacgcg tagtgcagac gcccccggcgctgaggctga 34620 ggaggcagat ggcccctccc cgcactgtgc agggcacccg gttgggggtggaggggaggg 34680 ccgcgtcggt gaagcgggaa agcctagtgg gaggattccc tggagctgaggagccggggc 34740 ctgggaaggg gcgcagaggc tccacccagg cgggggcggg aagggcggtgccagggcgga 34800 cagcggacgc gcgcgcctgc acggactcgg gcacacgcag cccttccgcggcagcgcccg 34860 ccgctccacc gtcgccatgg ctaccggctg gcctggagcg gggaggggcccttcctcccc 34920 ttcggcgcca acaggaggcg atttgagggg actcagcgtg actggtgcatcccggggttg 34980 gaaaatgggt gggtgcttgc gactgtccac gtgtggggga ccctggggttcgctttgcgg 35040 tagatgcaaa cgccgcggcg cgtgtgcggg gctctgcagt ggagcctgagccgtgccggc 35100 cgaggcgtgg tgtgggggag gctgccggcc ctctcgcgcg cggggtgttcacgcctagag 35160 cgctggggct gggggcctac cacccggtct cctcccagcc ccacctccgatttagctgtg 35220 tgaccttggg caggtgtcca gatgtctaga tctctctcag cccctggttgcccatgtacc 35280 tcataagggt ttggtaaaga tttaagtcat tttgtaaagc attttaacatagtacctgga 35340 acctagtaaa tgcttcataa atcttactgt ccctcatacc tgggtctcagttttcccagt 35400 tgttgatggg tttggagtga tcatgtgaca tcatctgagg agttgcccaggtcctcagcc 35460 tgagtgttga ggctgcggta gacccagctt ccgcgggtgc ccgtgggggaaggtggtaag 35520 tgtgccagtc tggctgatag atcagtttac accaggatgc ccagtgctcagccaggccag 35580 gcgcatggtg ggcgtcagga aagggctgct gtacttggct gagttgaatgttcagaggcg 35640 cctggatggg agagaaggaa gaggcagcag caagtcgctc ctgaggggctggagccctcc 35700 tgtaagaccc acttcccttc ccgggtggca gagtggcaga cttccgagtgctgcctagaa 35760 gcctagttgg cacaggggac tggcttttgg gtcccgctgt tttatggacagctctccaca 35820 cattctggtt ttaggctctg gcggcagtgc ctgagggatg atctgagccaaggacagagc 35880 cactgagggc gtgataattg agggaggaaa attaattgtc cttaatttggcgtaaatccc 35940 aaagaccttc ctcgtgtaag gaattcagag tagattccga gacacagggctgcacacatt 36000 tgtacttccc ttcccttccc tatctgcggg tggagatgaa ggccacttgactcctgggcc 36060 ctgactctgg caggccatgg ccacgtcttc cccatgagct gggcaggtaggaatgaggtc 36120 ttgaaagagt tagactggtg ctctggaggg cacccaggat ggccctagcagccccgagtg 36180 tccccaggtg ttggggaggt gagccctgca cctctggtcc ccctcaggccttcctatgga 36240 agcaagcagc agctgggcca aaggaggctg atcccctgcc tggtgcatctcagtcctctt 36300 catttccgtc ctccttccct tctcttgcca ctgttgaaca ttttacttttaaaaatctga 36360 aagggcactg tgggatcata tttacagcca aggagacact gggtttatatccagaacttc 36420 tagggctgca ggtggggaaa cgtacagcca ggtcccagga aaggctggggcggcaaggcc 36480 gtgctgggaa tcctattgct ctctagcctg agacctctgc tcctcatgggccacagtccc 36540 tttgggatgt ccctggcagc tagagccttg gggagcatcc cctgggactggcagcagata 36600 gataggtatc ttgctcctgc ctgttggggc tcgtccaact ccctcttctacccgcccccc 36660 agtcgctctc cctgggtctc caagaggctc cagggagggc tagtttctgccagcctttac 36720 cttcttcatg tctgaggatg ccatgtgcct ttactctggc atagaagcctgacttccctt 36780 ggcacatgtt cccatacacc catcttgtgc tgggcttgtg gaaaggaggtagagtggtgc 36840 tggtctcccc caccatgagc ccagctcccc gccttcccca ggagacagacaaagaacaca 36900 cattcccctt gccccacatt gggtgtgtct ggcatccaca ctgggagagacactctgctg 36960 aggccttgaa aattggtggt ttgggatggg gcctggtggc tcacagctgtaatcccagca 37020 ctttgggagg ccgaggcggg tagatcacca gaggtcagaa gttcgagcccagcctggcca 37080 acatggtgaa accccgtctc tactaaaaat acaaaaattt gccgggcgtggtggtgggcg 37140 cttgtaatcc cagctactcg ggaggctgag acaggagaat tgcttgaacccaggaggcgg 37200 aggttgcggt gagctgagat cgcaccactg cactccagcc tgggcagcagagtgagactc 37260 catctcaaaa aaaaaaaaaa agaaaaagaa aattggtggt ttggtcctagtgggaagggc 37320 ctctcaccag cctagagtgg aaaagggagt tcccgactct agtctcaatacctgtctgcc 37380 ccagtggctc agcccttact agttacacca gctaatattc actgggagtgaattccgcat 37440 cagatgctcc gcaacacctt gcatgcatga tcagatacag acctcacagtagccttacca 37500 tcaaggtggg cacttgaacc ctgtgtacag ataaaggaga caaagaatgagtaacatgcc 37560 aggccctaaa gctagttcgt ggtgaacgtg ggagtcccat ttgatctatactagtcctga 37620 gcctgtcctg gacttgtgct tccaaggggt ggagagtaga tagccttgccctgcagcccc 37680 tcggggctga tatgggagcc catgtacagt gggagtgggt ttgctgctgacatctgttcc 37740 tcttactcta tgctagtgac tcctctgtgt gccgccaacc cctagcaagctgggagaagg 37800 cagccaggag ggagtttttt ctccccctac caactttttg tgtctttagagcttttttat 37860 ctcctttgcc tccacacact caggcatggt ctgcagccct gaccgtgactcccagggtca 37920 ggactaagtg agggagaaag ctcaaggtca aggctgcagt aacgaataggtcaaggtcag 37980 gatcggagtt agaaggggat cattggtagg gctgggggtg ccccgggtcagggctagaga 38040 ccaggcggtg acctggggct ctgccatgtg atagagctga aggctggatgaagggaacac 38100 tgtgtgtgcg gacaggggag agggggctgg acagcacaga ggccttcaggctgagctgtg 38160 gctgttggat gctgcgcgag ctccctgcta gccccccccc ccaccccctgcagccccagc 38220 attcatgcag tgctttctgc tgtgaccagc agagcattga ttctcgttcttctcagggcc 38280 tggagagata agtgctgacg ccttcagtct gaggcgttgc ctctcagacctggaaactcc 38340 ctgacagggc aggggtgggc cccactgcag cctctgccct gccaaagagacttaggaccc 38400 tggttcctca aatcggggta tgcttcatgc ttagaagtca aggaaaggggaggggagtct 38460 tgagggccct ggccaacctg cagttgggga ggttaccccc agaggggtcatagggggcag 38520 gcagagccag ccctaataca cacattgctg tttgtctgca gaggaacagctgccccctgg 38580 gttcccttcc atcgacatgg ggcctcagct gaaggtggtg gagaaggcacgcacagccac 38640 catgctatgt gccgcaggcg gaaatccaga ccctgagatt tcttggttcaaggacttcct 38700 tcctgtagac cctgccacga gcaacggccg catcaagcag ctgcgttcaggtgagcagag 38760 ggcaggggtc aaggggccat gcagacctca gaacaagcgt cttgtcagatcccagcacag 38820 cctactccct tgggcctggg cacctccagg gctgagcgga gggtacctggtggggtgggc 38880 tgggtcttac tgcaggtgtg cctggctcag ggaagagagc tcgtggttggctgtgccgtt 38940 accttcttcg gattgtcaga ctccagactt tgggccagtt ctgcccctcccagcacatgt 39000 gatgtgccag tgtggtggac tcttcaaggg tgctctatgg atgttcaccctcctccttcc 39060 ctgtagcctg gcctgagaca gggcctggat gatgcttctc tttgcttcctcagatggcag 39120 ggcttagctg ggaaaagagg ctaaaggtgc ctgattcatc aggcttcaaaaggctggatc 39180 tcaggggcct ggaactaagg ggacttgctg ttgtccctcg accaccagagccacctgtct 39240 cctctggatg tctccgtcga gccagctcgg agccctggga gcaaggatgccatcgtgcag 39300 gagggaggtg tcaccccatt gatcgatctg cctgtgaggc tcctgccaggataattgatt 39360 cagtttttgt gggaacagag caggcgggaa aagaggctca gaatcagcttggctgcattc 39420 tgcatctgct gccagcacgg cctggaccaa tagtctttgc ttcagaagccctcctgctag 39480 ctatgggatg gttggcctcg ggcaggatgg ccagtgccgg ccagagccccttctgcctgt 39540 cagttgtgat gtcaatgatg aaaaggagga catgactctt gcccctttcaggggcctgca 39600 ggcttcagag gtgccctccc actccctggg atgccagccc ctccccatcaattcccacca 39660 gcctcacagc cccttggtgc ccagcaggag gagggagaga caagctgcccagcagggcaa 39720 gattctggcc ccagccacgg ccgcctgaga cagcccacga agtgttagctcatttaattt 39780 aattaaaact caacaagatg gaggcagctg tagcgcagtt aattaaaacagccataatca 39840 aggcagcaaa cggccggcag tgtttgtggc cgctgcccag cgcagcacagcggccagcac 39900 ggttcggctc ctctgcattt tctcatagtt cctccaggca ggctccccaagcagccagac 39960 gctcctccct gctggcctgg gcccctccac agaaccacat ggacttgtctggcagcagct 40020 ctgggaaggc tcgctcacac attggttcat ctagtattta tatagtgcttggggtgcccg 40080 gtcctgggcc atcccttttc ttgcctatca ctccactgga tgccactcaggccccatgct 40140 acttgagcta tgggtaggat ctaagattgc tgccttctta tcaaccacactgccgttttt 40200 agctagactt tgaaggactc tgctgttgca aataggctgt tagctatagctatatcctgt 40260 atttaatgtt atataatagg aaattatatc ttaatcctat aaaactaactttttatggtc 40320 taaagtaata aatgaaataa gattaataaa attaattata aaagatatgacttccctctt 40380 cctagtgccc cttccctgca gcagcatctt cccttccacc aagccctgcctttgccctat 40440 ggacacagcc ttgccctgga cgagctcacc ccctcctaaa gctccaactgccatctcctc 40500 acctgcaact ctagctttag tctttccagc ccaaacctct tacctgagctctagatccaa 40560 aattcgacct gccttctggt caccctgacc agtggtgatc agtgatgagtgatgaccttc 40620 ttttcagaca gctgtaccct ctttccataa gtggcaccct ccaacctggaacatgggtct 40680 caagttggtt tgagagctct caaatgtacc accctcgtgg ctctcaaattggcccccttc 40740 tcccctcctg ctgcgtcagt cctatcccag gcaccagaca ctgctttccctgcctccggg 40800 ccctcccgag aatccatcat cctgtaggtc aggtttgctg ctcataccttcctgtgcctc 40860 agtggtgtct ccttgcctac ctggtcaagt gacgctccca agcaaggcttagagggccct 40920 tcttggtctt cccctgcccg tgtctcatcg ggtcctggct acaccacttaccagctctct 40980 ggcattggtt aactttttcg tgtgtcagtt ttctcatgtt tgaaaaggagttacagtaag 41040 cagtgagtga gtcaatggca gtcaaacctt gagttgatgg cctggcctctggtaagggct 41100 tcatggagct ctttctactt tctgtaccct tgaccctcct aacactgagctgtgctgacc 41160 cgtccttggt ccttgcccac ctccggccct ctgctcacct tccggctgctccacttgaaa 41220 acctccaccc tgctcagcct ctggctgctg cctcaggcct caccttgctttacattgtca 41280 ccatctggct ccagttctgc tcagctaggc cttgcaggga gggcctgggtctgatcacac 41340 ttggtgaggt cagctgtagg acaggtcttc tctgagccct tgtcaagtgaatgatttcat 41400 gaacttgacc tttggcactt gtccctgtag gctaatatct gctctaatgttcactcctcc 41460 ttctgctttc caggtccaag gaagacttcc ttatttgtct tcctgccacctggaagttgt 41520 gacctcaggg tcgtgggccc agggtccagc tcctgggatg gagccagatggcacctaagg 41580 ggcctcaacc atcccacctc tgcagtaata actgggctct ctcccctccctgcctgacct 41640 ggcctgggac cgttggcctc agttgttgct ggccttatcc cattaccatttagaagggtg 41700 ctaaggctat tccgtgcaca tttttcaggg acagcccttc atggagtggactcaggcccc 41760 tgagcactca gctgtttacc gggaccttta cggtttacgc atcaccgacagtttacaaca 41820 gagctttccc cgcttttgtt gcagctgatt ctcttgcagc cctgtgaagtaggacaggct 41880 gtgattacca tgcccccttc acagctgagt taagggagtc acttggggtcacacatcgag 41940 actcggccta ggagctcccc tgtgagatca cctcaggacc tagtatcacaatagcaaacc 42000 tggggacctg aggagcagct ggacccttct ggggcttcag agctgcacattcccagcttc 42060 tccagacccc aggcccccac tgaccagtac ccagaagtcc tccaccatctgcaacctgag 42120 ccacagcaca tctaaccagg gcatgacccc ccaagtagga gctggacaggaggtagctga 42180 cggcatgcgc tgcccagatg tgagctctgc tcagcaggcc ttttcttctctgtagtgatg 42240 tgacatgctg ccaaaaccac ctcctggaga attggaactt gagaccggggtaggcccagg 42300 aggaacagga acaagcttat agagtggaaa tggagttgtg agcaggggctcagagcccct 42360 gctgggtcct gagaggagct gttggcctgc aggctgtgcc gagcctggacagggcttgag 42420 gagattcccg cactcctgct gtggcctgaa catatgagct gccatcctttgtcgtagagg 42480 acagcctaac tcactaagtc catgtgctca tccagagagc agtctcttccccacccccag 42540 caccctgagg cgaaacctgg gggtcttaag aagagatgca gcatctggctgcaggaggag 42600 gcccgtgggt gggacgcaga gggcttgcag cccctcaccc tgctggctggccccagctct 42660 ggctggaaga gcctgtcccc accccactct gctctgccat ctgcggggcctgccaggaag 42720 gcacactgcc agtgcatgct cacaatttcc ctttggccca gagctccctggcacctcttg 42780 gacacgaata cacccctaag gatgctgact tctgggcccc ttcagtcccccacacccatc 42840 ttgtgaaatg gaaaagtcag attctctgtt tggtggggaa attactgttagattctttca 42900 gaataggtta ggttctggaa gagctgaggc caggagcgag ggatgccagccctggaccat 42960 atccactgct cccaccccca ccaagtcctg gcgtggatga caggagatcagcaatgtcaa 43020 ctttttggtc tcaggacctc tacttgtaaa tatcagagaa cctcaaagagggtttgcttg 43080 tgtgggttct tttttttttt tttttttttt tttttttttt tttttgagacagagtttcgc 43140 tcttggtacc taggctggag tgcaatggcg cgatctcggc tcatcgcaacctctgcctcc 43200 caggttcaag caattctcct gcctcatact cctgagtagc tgggattacaagcatgagtc 43260 accacgcctg ggtaattttg tatttttagt agagacaggg tttcaccatgttggtcagac 43320 tggtcttgaa ctccgaacct cgggtgatcc gcccgcctca gcctcccaaagtgctgggat 43380 tacaggggtg agccaccgtg cccagccatt gtgtgggtta tttctattgatgtttaccat 43440 attagatatt aagactgaga ttattttaaa atatttatta atgcattttaaaataataaa 43500 aacctattat atattagcat aaataacatt tttaaggaaa aatagttatttttttaaaac 43560 aaaaatattt accaagaggg acaacattgg tttaccctta ttacaaatctctttaatttg 43620 ggacttatat agaaggcagc tggattttct agctactttt gcattagggcaattgtgata 43680 gcacatgtca tgtagcctct gaaaaactct acgttcatga gagaaagtgtataaagcaaa 43740 taatttctta gtattatgaa aacagttttg accttgcaga cctccttatggacctcctga 43800 aagggtctct tggagagaga gaatcattgt gcggattctt cccacataaccctcactccc 43860 aggtcccagc ctttgccctc tcccctgcct ctgcatctgc aggactggcagcctcatcct 43920 gtgggcaggc tatgagttag atggaccttg tcaagtcctt ccacttcactcacaagctgg 43980 tgtgaccttg ggctttgagt aattccctga atgtctcttg gcctctgttttctcatttga 44040 acttagagat gaagaattcc tctaggtgct gttgtcaagt tccatgaaacggattggtgt 44100 tgatttttct gcacacttcc caatccccag acgtgggcag gaagcaggcagcagggcagg 44160 gtagacgtgg gctggacctg ggcccatctg tgggctcctc actcctctggggaaggcagt 44220 ggttgcacag ggggcttgag cctcagagaa ggtacctgtt ggggatgaaacccgctcaaa 44280 tctcctaata ggcctggata tggagggtga acgcctggcc ctctgggttaccaaccctca 44340 gggttatgag gtgctagaca ggaaaagggt gggccctgat ctctgcctctcgccagccag 44400 gccaaattgg ggaagttcag tagtccccca gtttttagca gggttgagtgggctttctag 44460 ggactctgtc tctgggctgt gagtttgctg agtcctgctg cccgttcctctcggctgggc 44520 tgcgtgttgt gggaggccca gtgtgctagt agcccatggc aagccctccctttagcctgt 44580 gtcctgtctg ccttaacacc aggctttgtt ttggtaagtg ctttgttttggaatcttacc 44640 caccggtggg atttgggtgg ccctgtgggg tggggttact acctcttttcttcaccttcc 44700 ccctccattg gccccagcca gtggggtctg caggagtcct gactcacctccctgattctc 44760 gtcaccccca gccccactga gccccctcct cctccccaga gcagagccagtgacctggga 44820 cacagcttca ctgacactca gtctggctct gcctgcttgc tcacacactgtccgtgtcgg 44880 cctaactcag gcctcaattt ctccatgtat ttaaggccct ttcttgtgttttattttacc 44940 tgatttggct ttgtgtggct ttccttcctt ttatactaat gcttctctctgatcttattt 45000 gcattcaaat tacctcgcca ggtggttcac caatcagagg taagaatgctgtccgtgcct 45060 ctcgccacac cacgccttgc cactgccgtc acctccactc catcccgtccagtctgtcac 45120 ctccccatcc ccatcccaac ctcttcagcc tcctcatctc cagctccagcacccccaaca 45180 ccaccggcca ccacacacat ccactctcag tcattccctc ctgtggactcatccattgca 45240 agtctgaatt gtgaagatac tcgccgggcc ccgcctagga cagcaagccctgctgcccag 45300 gccttccagg ccatccccac agaagggacc ccatcagctc ctactgtgaaacttagcctg 45360 tcccccggct tccctagaca gaagagcttc ccttgaggtt gaggagtgtgaccaagcctt 45420 gccttttctc ttgacagtct cttttggggg gacctcaatt attacttgactttcccttta 45480 gtatcccggc tctgtaccgt ggtagaatga ggatcatccc ttcagcctggcaaggattag 45540 gaggaatgtt cacttaagag ttctgtgtag gtgctaggat gacaacagggaggtaaacag 45600 gctcgtgcgt aactccatca cctgacctgt cagctcggag tccccgctaggctgcctcca 45660 cgcagcaggg ccctgcagcc taagagttaa aagcacagat tctggactcaggaagatcca 45720 ggttcaaatc tgacttacca cctatgtgat cttcagaaac tcagttcatctctcagaagt 45780 gtatttcctc tttaaattgg gtcacggcca cctgccgcac agggtcatgagaattagagg 45840 agaggaggat gtgtagtgtc tggcgcagag cagacacctg taaaatggtggcttgtctat 45900 tcacatcatt ttctctccag ttctctcagt gtctgggcac atctagaccttcagagctca 45960 ggaccaggat ggtctcaggc aagagcagct gccttcctgg gtgctatcttgcccatcacc 46020 cggaggctgg ctcttgcttc acctgggagc cccccaatcc ccctgcccatctactcagcc 46080 tgtggtgact ctttgttgtc cctgctcctg ggtcctggtg ttggttccagatttggggat 46140 ttctgtatgc aaataggcac ccagtctctg ttaggccccc tgaccactcttaggctcttg 46200 ttgcagagga acctcctatt ttctgggtca gaaatttcac ccaggaaccactttctgccc 46260 caagcctgtg gcaccagcca ccaggtctcc ctcaccccac ccacacccccctgcctcact 46320 caggtgacct tagggcagcc tcgacccctc agcggcctga acccgacctgcttgaaggaa 46380 cattttctgt actagtgtcc atccatccca tcagcatgac ctgttgggtgcccatcaggg 46440 ctgctcttaa aggagatggt atttggcatg tgcactgggc cctggtgctccctggcatct 46500 ggtggggagg gctgaggcca gcaccaaaga ggcaggctgg tcctgtgtcctgcattggtg 46560 gcctcccctc ttccttcacc tccagctgcc agccccctgg gggttcccagtccctcctgc 46620 tgccctgggt tagatggtgg aagagggaca tcaggtagtg aggtggctataggtcagacc 46680 cagggtggta cccctttggg ccccaaccgt gagagcatga cttggaactcttcctctggc 46740 tcctgtccag tccaggcagg gggggcccgg cagcttctgt ccatgtcctgtgtgggtgtg 46800 attgtccatc agcctgggcc ctgggaaggt gggggctgtc aggttaggctggctgttctg 46860 tgcaggaggt ggttttgtca gactgtgtcc tgtgggggtt cttaactgttggaccgtcct 46920 gtgtgggtgt aattgtctgt tggattgttc cagggagggg tgtgctgcctgtgcccgggg 46980 gtgtgtctgt cacagacagc ttgcccagtg tgctttcaca agcagttttttcaagtgttt 47040 attggtgggg ccaggggacg gccaagtcca gtggatccac ccccaccttgccgctctgcc 47100 ttggcctccc ccgccaggac ctgtcagaac tggcctcaag cccaagattcctccaccctt 47160 tctgagggtc cttttgctac ccaccccgat cctggctcct gtctgtccgtctgcggccag 47220 agcagcccct cagggcactg gcccgagtga gagctccaac ctccattagcctcttgaatt 47280 atgcaggagc cgtggtgggt cgaagcactt tagcaggacc gggctgaggagtggagctgg 47340 aatgggtggg gctggcgggg agggggtgga gtaggagggg aagatggtgctgggagcaga 47400 ggctgctggg caccaggccc ccagggcaaa cccattcctt cctggtgggcggcaaattct 47460 caagctccag cctgatgagg gcatggcgtg cttgtgaatc ccaggcccgccagtctgcag 47520 gctgctccca cacctggctc tgactggctt agtggggctg cggggccaggaacactaaca 47580 gggatgttga ttgggaaata ggctccagta ctggcaagag tgtggcagaaactgaatgga 47640 gctgggggtt ggggtacagt ccctggcaga gcaaaggtgg gtcttgctggactgtgggga 47700 gcatgagatt gtgctccaaa gcacaaccgt tggttctaga caggaggccactgggctctt 47760 cagggagagc ttcctggaag cagcaggggt gccagggtgt ggcctggatgagcttcgaca 47820 tctggtcaac atcagccaca gatgctgtcg agacctctca ccgtgttccaggccacactc 47880 gacatgggcc gtgcctgacc tctcgcttcg tgtacccaca ggtgccttgcagatagagag 47940 cagtgaggaa tccgaccaag gcaagtacga gtgtgtggcg accaactcggcaggcacacg 48000 ttactcagcc cctgcgaacc tgtatgtgcg aggtaaggac tcaggcagtgcctggcccct 48060 gtcaccacag agctgtgctg cacctgccgg gctctctgcc cagagcccttggtgcagaca 48120 cgcaagggac tgccatgggc ccagtctctt ctccttcctg cttctttctgcagcagcagc 48180 aacagctccc actgggcaag ttcctggcgt ctgccactac ttcgccttccttccttgcag 48240 gcccatgggg aagcagccac tcttgggagc atttgtatct tttgtaggtcttgccgcatg 48300 ggcccggagc cccatgggaa tttggagcca tccaatccga cttcttggtgtatgtgcatg 48360 tgtgtgtgca cacacgggca cacttatctg tgtgtaaatg catgcatacctggcctggct 48420 ttccttcagt acatcctcct gcctgccgca gcatggtggg ctccagaaccaccatggctc 48480 tgggctttgg ggtagtaagg cctcaggaac ggaccaggcc aggtgagcctatcctctggc 48540 caggtcttcc tggagccttc ctggaggaac ccttgtgttc agctgtgagcccctgtggtt 48600 atgttgcctc gggtgagcac ctggtgtgtt ccagatgttc tttgctgggagcgagtggac 48660 agtgtgcttt ctggaagtcc aagttcccag cagagaggaa cctggggactgttttggggt 48720 ttccaatctc tgctatgctc acagcctagg ggtgactaaa gctggccaccttgccctttc 48780 ctgagaacac actgggtctg gtatacatgt gcccagaagg ggccctgagagccccttcta 48840 ccctcatact gtcccctcac ctagccttct tgtacagatg gatgtcattaaaagttaaac 48900 tgttgaccgg gtgtggtggc tcacgcctgt aatcccagca ctttgggaggctgaggcagg 48960 cagatcacga ggtcaggaga tcgagaccat cctggccaac atggtgaaaccccgtctcta 49020 ctaaaactac aaaaattagc tgggcgtggt ggcgcgtgcc tgtaatcccagctactcagg 49080 aggctgaggt aggaaaatcg cttgaaccag aggttgcagt gagccaaggtcgcgccactg 49140 cactccagct tggtgaaaga gcaagactcc atctcaaaaa aaaaaaaaaagtacaactgt 49200 ctccacccag gggattgtaa aggaaagcgg acatggctcc ttgcagtgatgattccatca 49260 cgccaagtgc ttagcgcggc ttcctgaggg gtgtcgtctg actctccacccagtgcacgt 49320 ggtgggtgat ggcggtggcc tgtccgactc tttggggcca tgtggtcagacttgcttact 49380 cagagcagag agaattaggc tgtcaaggct gtcaaccaaa atacacttatcaaggagttg 49440 ctgtctgtca tgggagaggg tggttctgga gaggccacag gctgccagcccattcggtga 49500 ccagttgagc tgcagaggaa ctatttgtgg tgggcaaatt gtgcttcagagactattaaa 49560 gatgcttcat gagagttggg ggcatgggat gggaggtccc ctagcttactgtcaggtgtc 49620 ttactcctca aggctcaggc ctcaggtcag cttgtctgtc ttcaggaacaagagcctgaa 49680 aacttcccaa atggcctaga ggagtggagt tgtaagcggc acagccttggccccaccctg 49740 ccccaggcag agtgtctggc aggcattggg gctggcgagt ccactccaacagaggagcag 49800 ctggaatgac ccttctcaaa gatctgcctc ctcccaccct gactatcgcaaatgctgtgc 49860 tctctagagc tctgctcaca agactgtagg gtcgagagtg gcagttccaggcctcagcaa 49920 gaaagccttt gcatgtggct gcaccaggga gaggagaggc ccagtagagccactgtaaac 49980 ccaagccagc tgcaagggcg tggcctatgg gcacatttcc cagggcacagctacaggagg 50040 cagtgctgag gcagaagaag gcccagaggt ggatggaccc ccgcattgtcctcttgacta 50100 ggcagcattc tctgaagtca ctgttacact ggccccagcc ctggaggcagagcagtgggt 50160 ctctttggag atcgactgag ctcagctgct gtagctggca gctcagtcactgcccaggga 50220 gctcttatgt gcagcccctt ggagcatccc tgtctgtggg gctccctgtttacacatggt 50280 agactcaggc agggccctga ctggctcttg ccatggccca acccctaactctaccagctc 50340 ttcttgaatc tacccttctg gctccatccc cacatgcctt ttggtaggaaatacgggggt 50400 tcctttagtt ccagccccac ttgccaggga ctccaaaaca gccaaacctgacttctgttg 50460 tctctctcac acacacatac gacgttcctc taggccctga tgtctcaggaccgtttctaa 50520 gctctgtccc caggacctgc ctcatgtaga ccctaattga atctacaccaccctgctttg 50580 ccaagagcaa cctgccccat aggccctact gtcctactga caggacttaggcacacctga 50640 aaggccctgc tccctgggtg ccttgcctct ggtgccccca ctgctcgtcatctcatggtt 50700 ggcaaaaccc ctgccttctg ccctcagcag tactaggggc ccttcagccccagcgcctgg 50760 ggacagcctg tactgggctg accaccagct ctgcatcctg agccctgcttgtgggtggat 50820 ggccgtggag gagtccccag tgccagcctg ctttctggcc tgcaccccgtcccatctccc 50880 agcagagagg cttcgaggtt tcctcccaca gcaggctcct gagcctcagacggcttgatg 50940 tgggctctag ccctgaactt ctcagccgca gcactgttga cattttgggctggacgattc 51000 ttagttgtga ggggccatcc tgtgcattgg agggtgttta gcagcatctctagctcacta 51060 gatgccagtg gcacccctca gtcaaaacaa acgttgcaac gtgtcacctgggggaaatta 51120 ctgatggctt cccactgcca ttgtctccac ccatctgact ctcttcacctgtgtccactg 51180 gccgcaaggc ttcgtctcct catcagactt ggcttcggtg tctacctcacatctcccttc 51240 tgtcccccaa attctcaggg ctcccagtgt cgctctctga ggccacgcgactgctacatt 51300 taatgggaca gtacacgtga agcaccaggc acattgccag gcacacaggaagcacttgct 51360 agtcagtagc ctctgcagct agcactcggc tactagtcag tagcctctcggatagcactg 51420 tggggggatg tgtcatccag ttacatctga ctttgttcac agttgcctgcagctccaccc 51480 acagtctagc aggcccaggc atgctgggtg ggccagagcc tttctccttcaccacctgac 51540 tctccctgag tgactcattc tctccttcca tctacagctc tctgggtgtacagctgctgg 51600 ggccagggtg gagccctgcc ctcctcagag cctggcctac ctgtgccaggactaccagcc 51660 ttcccccttt ctctagggac ctggctgcgg gccacagctg tctaaaacagggacagtgcc 51720 tttttcccca caggtgccca gacatgctcc ttacaccggt ggtgtgtgtgggggtggctt 51780 ctagtggctc ctgtaccttg gcaggtttgt gggctgggtg ggccttgaccccagagcccg 51840 gtccacaggg tctgtctgag ctgtggggtg cgtgtgaggc atgggggcctgcctgtgccc 51900 cattttcacc tgccccggcc ccaccctcgg cctccctggc gcctgctggcgggcctcagc 51960 cctgtccacc atgtcctcca tgagtcctga gtcttttgtg agtgatgtggttcgtgtgca 52020 cctgtgtgca tgtgtgtgtg cgagggggca caggagtctc gtctcgtctccgtgctgtgt 52080 gggcagatga aggttggcct gtttttactc tctctgtgtt tctccttgtcttttttttat 52140 tccctcctca tcttcatcgc actctgccat caacccaaac tctcatctctcagatcagcg 52200 agaaggttgg tccttttcac ttcttatcca tctacagttc gcccatcgatgggacacgcc 52260 tgtcagtagg gcccagcggg ctcggtcagc tccctcaggc tcactgcgccgtgcctgcct 52320 gccagtctct gttgtttggg ccggcgggca ggcaggacca gggatgggtgggcagacccc 52380 tgacctgcat gttcctgctg cttgggtccc tggtgcacca cgtgtctgcatgtcccctta 52440 gcctggttcc cctgcaaggg tggtggggtg ggcatgctgc atggcatgaaactgtacccc 52500 acctttgcac ccaggccggg ccagctgtct gatccaggcg gtgctcaggaatggttgtgg 52560 ggggctgtgg tcagagagag gtgtaagacc tgtagcgctt tgtgtgcacctgtgggacct 52620 ttcaccgccc tcctcgccta gagacccagg ctcagccgaa actactggaacattaaggcg 52680 agaggctaga gcaggctgtc ctcccagtat cctgcagcac agggcaaaagtcctgcagtg 52740 tgtaccctgt ccttgaggcc ctcaggtccc ccaccagggc ttaatggggcagtcagccct 52800 ggtctgcctc agggagcttg gtgtccatgg cgcagggtga gtagcgtcatcttggcaccc 52860 tgaggaagtc tcccataagg ttgtcttttc cttctccagc cagatccaaattaagatcct 52920 cctacctgcc ccaggcccct ctgcccaagc ttcccagcta gctcccgtgtctgtttgagg 52980 gtggaattta gcccaccagc cagctaactg acattcctta gcttgatgctttgcattcag 53040 actcaagttg cttctacctg gagtaactga acggtcagct gcaacttagcaggatccata 53100 tgggagcctg gaagtggccc ctgactcagc cacaggttag gaagggtttcctggagactg 53160 tccttgagcc cattccagag caggtctgtt tggggcttgg ttttccgccccacccttgga 53220 gccagacccc caccactgat gctggggttg gcagcagggg ctagtggtcattcatgtatc 53280 acatggtctg tgtggtcaga ggagactcac ctgtccattg ggttttgggggttcaggata 53340 gcagtgtagg acaggctgag cctcagggag caaacccagg ccatcctcgttccctctgcc 53400 acatcctccg tggcttgagt ttcctctgtg agggtgaggg agccggtcgagaggagggct 53460 atagcctagg ctcagatatc agccctgagc atccatccat atgcacagctgagcactggg 53520 gcatttctgt tacaccattt tgctgaccgg gcagatctgc gagggcgtccagaaaacagt 53580 catcagcaga gacagcccag cccagagtgg ctccttccat gccccttcctctaccctcct 53640 ggtccctcgt cccctccctt cccccacccc accctatggt cccaccaaaaataagcacat 53700 ggacaaatat ttagcgggaa ggcagacatg agtttctaaa tattccagggggatttgccg 53760 ggctggtaat ttgtttggtg ctgataattg catcttacat ttttccagctttacttaaaa 53820 ctgtgtgccg ggtttgccgg catttaatta ctgctctgcg gcagccacaaggttatttat 53880 taaagagtta ttttatcttg atacagtgga tcctgcccct catcccctccttaatgttgt 53940 gttattttca tcagagaaat ttctctgagt gaatgcagat tgcggggctgcctccccctg 54000 cgattaggct gtcactctac tgaatgctga tgcctctggg cgccgcaccgccctattata 54060 gggggttgtc agcgcaaata attagactta aaagttacag gtgaaatataatccagaaat 54120 ggcagggccc tgttttggaa attgtctata aaatgtcagc actaaggatgcacagggaac 54180 ggtaatatac tggcattgtt ggagacctaa gattaggacc tgaagtggcatttttcccag 54240 ctccagtttt tctttccctg ctgttgcttc tgtcaataga ctgtccagggtgaggcctgt 54300 tcctttttgg aggccctgtg cctgcagcag tgggtggagg atggttggggctgctgcagt 54360 gagatgccag ccactccagc tgttggcatg ggggtggctg tctgggcaggtctaggatgc 54420 gggtccctgt tggcacttgg aatacaagga ggttgtccct tgcctgtttcccccaccctt 54480 cctctgtaga cataccctga cctccctcac atggaaaatg tctcagtctcagtaacgttt 54540 ctgcgtggct gaagcccagt gtccttgtga gagtgaaggt ggtgtgcccaggagcctctg 54600 gacctgaggc ccttgcttgg tgttgcaggt gaccctaaag gccactgttgacctctcaca 54660 cttatgccta gcccctagag ctctccctgc cccctgcatc taggagggaaaaaataaaca 54720 caactcttct gtgaagcgcc tcaatttcct gacagccgca ggagttggaggctttagaag 54780 acgtggctca gggcgttggc ctgacctcca gggctgatcc atgaagtccgccttttggcc 54840 tgaagaccct gtcctccagg gacaatttgg gggtaaagtc cagtcaagtttctaggttgg 54900 tgcccaaggt ttattggcca aggacctact tccttctgct ggggctctttgtcctggtat 54960 ccaacgacgt gattaggccc catgattcca ccaccgactg gggtcaagcctgcaatcatg 55020 acctctccct ggcacaggct gtctaaaccc atccttacgc ccgctggagcagcgtcctct 55080 cctgccagcc tctgctaatt ctgatgcttc tccccagagc cttctgggtcttttttcagg 55140 cctgtcagct agggctcgaa agggtgacag tttttcatca cacagacctactcctactac 55200 gtatataaac ttggtcatgt ggcttggttt taaaacagtt cccttgactgtaattggcag 55260 gtgatagcat ctacagcctg gaaacctgca cctagtaggt gcttaagcatagcttccctt 55320 cccatgaaga ggaggagaca gggcacccac tggccgagag gacaggagagatttagttca 55380 ttaaggctgc tcctctgtgc tgcccccacc ccgcttctgt ggcaggcctggacctgatct 55440 gcaaacagac ttgcctgcct gtgcctgtcc ctgaggccca tctccagagcagagggaggg 55500 catgcaccgc tgggctgggt ggtggtcctg gctgagcctc ctgctctcaccttggaccat 55560 ttgaggtgcg tgctcagaga gtcctttgtt gccatgagac acttgccagccatgccccgg 55620 gatccacctg ttctgccaca agaactgtgt ctgtgacatg ctgtcatcattggtagagac 55680 tcctggcact agaacagatg acagaaaccc acttatacca gcgtaagcaaaacaaggaag 55740 tcctgtgtta caaatctgaa aagtgtagta gatttggtgc tgggcagggctagatccagg 55800 tacttaaaca ggttggccag tcatttgtct gtctgtttct gagctctcctctcctctgtc 55860 gacatctttg ttctcaagca gcttttcctg tgtgttggga gaagccatccccagctgctc 55920 taagcttagc tacctcagta gagagagaag gtcgcttttc taatagtttctgcggaagtc 55980 caaaggctgt ttctccatag tcagatctgg gtcacacacc ccatgtatgaagaatgcagg 56040 tagaagtggt ttccttaaag acaatgggaa tgctcattga tttgaggagaaagcagaacc 56100 ctgaatgtct acaagatact ctcatgccct aggtgaacct ggggacacagatccagccca 56160 ccagcacctg tcaggcctgg tctctggggc tgccagggtt tgggttctgtctggagcacc 56220 agagtaaggc aggctgtgga aggagttgaa gcaagtgcta aattaggtagggtctcttgg 56280 gctgggcaga ggagtcactg ggagccatgc aggagcttgt taaacagggagtggcatcag 56340 atttgtgcca gctgtttgtg gagggcccgt gagcaggaat tggggcagtggacaacaagg 56400 caggaggact gacgcagaac acattgcagc aactcaggga aggatgatgatggcttggac 56460 tttggtggtg gcagttgcct gtgtgtatgg agagaaatac atggatttggaagatatgtt 56520 atgccacgct gttgacagga cctggtgact tgctgaatgt ctgatctctgggtctaagtt 56580 ttgacccctg tatggtgagg tcagacgttg gaccgggagg tctctgaagttgcttctagc 56640 tctagttctg tgatttgggg ctgtgtgagc agcatagtct ggtgcttatagtggagatgc 56700 tccttatgtg gtgctgtggg gtctttgcat gcctgggccc cccagcgagggtcctcagag 56760 accagtgctc tcagcagtgc tcttgggtac tgactagcct gctgcctgccgcctttggcc 56820 ctgcgtgtga gagtgcccgt gtaggacaca ccctggcctc tgctcaccagctgcatggcc 56880 catctgcatg ccagacatgc ctgcccacgg ctgggtttca tcacctcccttccctctgca 56940 tgctcctgtg tctgtgtcaa gcttccaact tcgggggcgg gtggttctaggctgtaccct 57000 ggactgagaa gctcagaaca gcttctggtc aggggagtcc aaggctggaggtcgtggcca 57060 ggcacagcca cagccacccc tccccttgag acccttgttt gttggcgcaggaagccgtct 57120 gttcggcgcc ctcatcatgt tactgccatc gtcatgccca tcctgcccgcagaccatgtc 57180 cctggcctgc cctgacccct gcccctcccc agtcctcctg agggcagcccttttgcttgg 57240 gttcccaggt tgtgccctga gcatggctga gaccctggca ggcggccctgctgcttgcct 57300 tatttcttcc cctcaggcca tggcctggag gtggaggcag tgactccacggcaggtggct 57360 gtgtccctct ggactggcct tctgctctgc ccagccatgc tctgaggaccctccacccta 57420 gggcctgctt tctctccttg gtgctccagc caggagcagg cagagataggccctggagaa 57480 caccctgggt tgtggtcctg accaggcctg gaccttgtac tgagtccctgtgtgaccctg 57540 ggcagacagg accttcctga caggcctcag tttcctaggc tataaaatgggagcttggtt 57600 gcaagatctc tcattgagta agtcatcgtg ctccagacag tccctgagtgtgggggccac 57660 ttctaggctg gaagtgggtg gggtgggagt ggatgatgag ctggggtctggcggtgccac 57720 ccctcctgtc tctgagcatg ggtttggctc tgaccagagg ggcttcctgaatgaggggcc 57780 cctgcccttc catgcagtcg tgtgtcctgc ccggcctgtg agtgcctctctccctcctcc 57840 tgcagtgcgc cgcgtggctc ctcgtttctc catccctccc agcagccaggaggtgatgcc 57900 aggcggcagc gtgaacctga catgcgtggc agtgggtgca cccatgccctacgtgaagtg 57960 gatgatgggg gccgaggagc tcaccaagga ggatgagatg ccagttggccgcaacgtcct 58020 ggagctcagc aatgtcgtac gctctgccaa ctacacctgt gtggccatctcctcgctggg 58080 catgatcgag gccacagccc aggtcacagt gaaaggtgag tgtggcaggtgctgtaacca 58140 gtgccctccc tgtcatctgg gaggtcctgg tggtgggcga atgtgagctggctgccatgg 58200 gcacaggcat ggctgaggga ttcttgccct ttcctgggtg tccctgccctggggtcctcc 58260 agcccttaga gggagggagg gatttctgtt attagccggc ttaatgataatagttaaggc 58320 atattgagtt tcttggtggg agacatcatg ctagcaagca cagttgattttgtttttttc 58380 tgttttaccc tgggagatag gtgctcttat tatttccatt tttgaaacgagggaaccgag 58440 gcacagagag ggtgtatcac ttgccccagg gtcacataaa aataaatgacagagcaggga 58500 cttaaaccca gtgtggtctg aatccatatc tcaccctcac cactacatagtaccagacct 58560 tcaggtttaa tagcctccca gcaagaaggg tgtggtaaaa tagtaggggaaagtgtgttc 58620 tctctgccct gatacctggg gacaggcaca catgtattac acatatgtcacatatatgta 58680 accgattctg gccaggcacg gtggctcatt cctgtaagcc tagcgctttgggaggccaag 58740 ttggaaggat tgcttgagct caggagtttg agaccagccc agttaacgtagtgagacccc 58800 catctctacg aaaaaaaaaa aaaaaaaaaa actgggcatg gtggcacacacctgtggccc 58860 agctacttgg gagggctgag acaggaggat cacttgagcc caggagggtgaggctacagt 58920 gacgcagtga gccatgatca caccactgca ctccagcctg agtgacagagcgagacccta 58980 tgaaaaaaaa aaaaaccaga ttccttcctg gtctccccgc tgcaattctcaacagcctct 59040 gatccattat ctgttttgca gcttgagtga tctttaaaaa atgtaattcaggatcaagtg 59100 actccctttt aggaaaggga gaaacacctt ccagtggctt cccgttgttttaggatacag 59160 atcaggatcc tcactgtggt ctcccagggc cactggcttc ccaccctgtcacgcgccagc 59220 tgtccagaca catgctgctt tccgtttgcc gtgctccttg cctggccccttctgtcactg 59280 gctccatgta cccttccagg cctcagcttg gtgatgcttt ccttccctcatcaccgcatc 59340 caaggagatt ccctggatct tactccacct cagcaccctg attcctgctgtccttgcaca 59400 tggcagaatc atgagtacga tcgacttgtt tattgattct ctcttgtactgaggtgtaaa 59460 ctccatgaga gcagggatgt gactgtgtgg cattgtagcc ccagcacaatgtctggcatt 59520 gagtgggtgc ttgttaatta tttgttgaat gactgtggac tcaggacctttccacttcaa 59580 tttgagccaa gctgcagggt ctgtggggcc agcagcccca tcactctttctatccgggcc 59640 agtccctaag gaaatatctc cccttcccct gcctattaca catactttccaccaggtggg 59700 ctcaggtgac ctgcagaggc acatagctgc tacccagtct gggtgtctttcatctacatg 59760 ggactgcaag ggtcacatca cctccaggtc tgtttgttag atgagtctggtgtgctgatg 59820 ggagggtcca gtgaatccca cagttgatat gtgtacatac taaatgacccaaggccctgg 59880 gtggggcact gacaggcctg gccactgtcc ctcacttgtg ccttcagaggtcaccataag 59940 cttttccagc catttttcaa ggtaggctgt gggtatcagc cacactcaggtgaccccacc 60000 agatatcctg gataatcccc aagtctaggg ttggttccta aggatcttgacctcgggcag 60060 ctttgagcct tccactttgt ctccagctct tccaaagcct ccgattgatcttgtggtgac 60120 agagacaact gccaccagtg tcaccctcac ctgggactct gggaactcggagcctgtaac 60180 ctactatggc atccagtacc gcgcagcggg cacggagggc ccctttcaggaggtggatgg 60240 tgtggccacc acccgctaca gcattggcgg cctcagccct ttctcggaatatgccttccg 60300 cgtgctggcg gtgaacagca tcgggcgagg gccgcccagc gaggcagtgcgggcacgcac 60360 gggagaacag gcgccctcca gcccaccgcg ccgcgtgcag gcacgcatgctgagcgccag 60420 caccatgctg gtgcagtggg agcctcccga ggagcccaac ggcctggtgcggggataccg 60480 cgtctactat actccggact cccgccgccc cccgaacgcc tggcacaagcacaacaccga 60540 cgcggggctc ctcacgaccg tgggcagcct gctgcctggc atcacctacagcctgcgcgt 60600 gcttgccttc accgccgtgg gcgatggccc tcccagcccc accatccaggtcaagacgca 60660 gcagggaggt aggtgggggc atgccggctg ggcagccaac agcagagaaggggaggctga 60720 ggttgtggcg gtgcctttcc ccctccctcg gctgtgaggc tgggggctcttgggaggatc 60780 aaggtgccgt attccataga tgtgtggtca gttgggatgt aggataagggtgtgaggtta 60840 ggacctgact tcctcggctc cctcctccct gggcacccct gacctcacgcagatgaggct 60900 gacctgcctg gtgtggggtg ttgcagtgcc tgcccagccc gcggacttccaggccgaggt 60960 ggagtcggac accaggatcc agctctcgtg gctgctgccc cctcaggagcggatcatcat 61020 gtatgaactg gtgtactggg cggcagagga cgaagaccaa caggtgtgcagcgggcagag 61080 aagcactgag ggggtctcct ggtccctgag ggtctgtgat gggcttaacccaggagggta 61140 ttttctggat tctgtggctt atgtgggcac agcctctaag gtttctgctggaggcttagt 61200 ggtgcatgcg tgtcatgtgg cccgcacagc ctgtatgaat ctggagtcattgtcaccctg 61260 tgctaggggc aaagtcccca gggtctgcct tgggtttcct aggctctgtggctcatatga 61320 ctggcagagc cctgaagttt ccccagaagc catgcgtccc agatggctcctgtggtccat 61380 gtggtctgtg aggcgtctgt ggcatgggct aagccctgag ttccttttgtgctccatgtg 61440 gccttatggt gtggagccag tcctggagcc gtggtgggtc cagaggtgtcacattgcagc 61500 ccatgttggg gaacaacctg tgagtgactt tgagctcaga gctgtcagtgagtgctccct 61560 gctcttccca gcacaaggtg accttcgacc caacctcctc ctacacactagaggacctga 61620 agcctgacac actctaccgc ttccagctgg ctgcacgctc ggatatgggggtgggcgtct 61680 tcacccccac cattgaggcc cgcacagccc agtccagtaa gtgtctcccaagtccgctgc 61740 ctgttacacc tgggctggga cacacacaca cacatgcaca cacatccttccccctcgacc 61800 aggcaggtgg ggtggtcagg tctgctggcc aaaccgaact ctggcctgggctctgagtct 61860 gggcctcggg agagggccag gtaagtgcct cccagtctgt ccagtccaggtggctgctgc 61920 cctccctgct ccctgcccag cctcctgccc ctcctcccgc ccatgccccacacactgccc 61980 aggtaagttc tgtgtctgtg cctcatctcc gcccccactc tgtgctggtcacactagctt 62040 agctgatggg ccatcctcgg tgggtctgcc atgtcccaga cctgctttggcggctgtttc 62100 tcctccctat ggccccgcat ctcgtctccc catcacccac attcccttcctcatactccc 62160 catgaaagtt acctgagagc catgtgatca ccctgagctg actgcactcggggacaggtc 62220 tgagaatgcg gccatgtgac ccagcctgcc cacgtgtgcc ttgaattgtgaccacgtggc 62280 cgtgcagggg actgtctgca aggctgtctg tgtgagactc tggtgttgtgtgtgagagac 62340 tccgatgctg cagctgtggg agtggcactg cccatgagtg tgagtgtgtgaccacacctg 62400 accagctggc tgtgcagcac ctgtgtatgt gcatcagtgg atcttgggcatgacccttgt 62460 ctttgtgggc gactctggcc tgatcctgta accgtatttg tgggtctcggtagcccccca 62520 gacagtagac tgtgtgtgtg tgtgactctg tggttgctgc tgggcgtcctgtgtgtggtc 62580 acacagccac agtcagctgc tgcgtgagac tctgtgtgat agggtgtcatggatgtgact 62640 ggctgggtag cattgtggga ttgagatggt gaccacatct gatgtggccatgtgatgacc 62700 agaaacatgg cggtgtctga agtggtcctt atgaagtggg accatgtgcatgtaccagtg 62760 atgtcaagag gctgtgtgtg accttgtatt catggattca ttccagaagcacctgctgtg 62820 ctgcagctgg gggccaggcc ccatgcacgg tacagggctg gcagcatgaccagacacatg 62880 gtgtctgccc atggggatcc tgcattccag cagggcagac agacattgcacctgtaatta 62940 cccaaatgag cttttttatt ttatttattt ttcaagacgc agttttgctcgtcacccagg 63000 ctgcagtaca atggcaccat ctcggctcac tgcaacctct gcctgctggggtcaagtgat 63060 tctcctgcct cagcctcccg agtatgtggg attgcaggca accaccaccacgcctaaaaa 63120 gctcatttgg gtaattacag gtgcaatgtc tgtctgccct gctggaatgcaggatcccca 63180 tgggcagaca ccatgtgtct ggtcatgctg ccagccctgt accgtgcatggggcctggcc 63240 cccagctgca gcacagcagg tgcttctgga atgaatccat gaatacaaggtcacacacag 63300 cctcttgaca tcactggtac atgcacatgt accctgctcc tgttcatttaccagttggct 63360 cccacccttc cttcaggcct tgggaaagtg tcacctcccc aaggcaaccctctttgaccc 63420 ctcagatcaa cagtgaaact ccgtctcaaa aaaaaaaaaa nnnnnnnnnnnnnnnnnnnn 63480 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn 63540 nnnnnnnnnn nnnnnnnnnn gtgtaatccc aaatactcgg gaggctgaggcaggagaatt 63600 ggttgactct ggaaggcgga aattgtggtg agccgagatt gcgccattgcactccaacct 63660 gggcagcaac agtgaaactc cgtctcaaaa aaaaaaaaaa ggtaggatggaagagacagg 63720 gtggagagat gatgagacct cctttgatct gaggggtcaa agagggttgccttggggagg 63780 tgacactttc ccaaggcctg aaggaagggt gggagccaac tggtaaatgaacaggagcag 63840 ggagcacaca ggcctccggg agagtctgca ggcctgaagg ccttccagctgcaaggcaca 63900 tggttatggt cacaaggtaa gcagcaggcc agtgtggctg cagtagctgccggatctatg 63960 gcagtcaggg ccttggggac acagggtagg caggagagct gactctggctgctctgtgag 64020 aatggacagg gcagcctgtg aggaagcaga gaccggttag gaggctttgtagcggtccag 64080 ggaaaagatg gtggcgcagg tgaggtgatg gcggtggatc tgagaactgtcagggatgct 64140 gactggtcag acttggtaat gggttatgaa taggggccca gggggagggaagtggaggtg 64200 aatcttgttt ctggtgtatt aacctgggag gagagtggtg ctggcgtagaatgggaaatg 64260 gggcagaggc aggtgtgaaa actacagctg gtctcagaca cccaagttcaagatgccatg 64320 tgccatctac atggcagtgc tcggcaggct gtgggaatcc agggctgggctggagctaaa 64380 ggaccctgag aagaaagact gcagatgaga ggaaatcagg ggaggggagcgacttggagg 64440 ttggaaggag agaaagtttc aagaactaga cgagatgctg ctgagcggtcaggagtacct 64500 gttggaccga gcaacatgga cgagtggccc tggagatgaa gcttggtgcatggtaggacc 64560 aaagctggtg gatgtttcat gaggcctgtg tgggtgctga gaatgtggaggcagcaagcc 64620 tagacgttgc cagaaagtgt agctctgaac aaggggacca ctatggctagagagggccgt 64680 ggagctgagg gtgggatttt gttttgtttt gttttgtttt gtttttgtttttttgagaca 64740 aagtgttgct ctgtctccca ggctggagtg cagtggcatg atcttggctcactgcaacct 64800 ccgtccacct cctgggttca ggtgattctc ctgcctcagc ctcccgagtagctgggttta 64860 taggcgtgcc cgccaccaca cccagctaat tttttttttg taattttagtagagacagtg 64920 tttcaccgtg ttagccaggg tggtctggat ctcctgacct cgtgatccgcccgcctcgac 64980 ctcccaaggt gctgggatta caggcgtgag ccaccgcgcc cggcctgggatttttctttt 65040 aagatgggaa aatttcaagc atatttaaat gctattggaa acagtcaataggaaaaaaag 65100 aaaccgtgga aaagagggta actggtggag ccttgggagg tggagagaaggggaacagta 65160 tgggtggtga gagagcttag ccttggacag ggtggctcaa ctctccattctcatgggtgg 65220 aaaggagaga attgctgcag atgcaagtac ggtggggggg ttgtagcaagagatggaggg 65280 agctgctgtg aaagcctggt cttctctgca gtgtgggaga catggtcatgcgccaagagg 65340 aggcaggcaa agggagctgg aggtctgggg agcatggagg agggggactgtttcatgtga 65400 ggtggcagag cgagccaggc agggggcaga aacaggcagc atggagagtcctggaggatg 65460 aggcctctag ggacgtgtgc ataggacgcc agcctgcctc agtgccgctgtagagaagga 65520 gggggagagc tggattcatc tagggcttgg cagcggccag gaaggagcatggaaagatca 65580 ggaggcgagg gaccttgcga tattggtgag agtgtggtag gtggtagactgtggaagctg 65640 aacatggagg gtgggaagga caaggttgat ggacaggcat cctgaggagctggcgagcag 65700 gctcagtgcc cggggcctgg agtgactgag caagaacaag ggggtggggtctgcaagggg 65760 atgtgagagc ccgtgattct ggagggaggg ccatccccag tactggcagcctccagggca 65820 tgaccacagc tgtgcagggg aggagaaagt cctggagatg aggggctctggggccagtgg 65880 gtcaaggggc tgctgaagtc cccatggcag ggggtggtga cagaacttgaagaggaaacc 65940 tgtgcattgc agcagagtcc tcggtgggcg tggagtctgg acacatcattggatacaagg 66000 agacacgtgt ctgcctccta attcccaaag aagctgttga gtgagggcacctctgttggt 66060 gggtactttt tctccacgcc aggggctgtt ggccttcatg ttcggcctcctgttggtgat 66120 tacctgtggc actggcaaag agactgtttc caggcaggca caggtgaaggctgccttatg 66180 ccaaaagggc cactggggcc actcttcctc ggaagagcag aaccgtgggcacagcagatg 66240 tgaagcagtt ctccatatgt ggctgtgtgt gcgggtggca gtgggatggccacgtgcttg 66300 tgtgtatgtg atatcgtgta tgttttgtgt gagacagcat atgtgtgggagagacctcgt 66360 gtgagagatg ctgtgtcaga actctaagac attgtgtgtg agagtgtgtgtcagatgcca 66420 tgtgtgagac accaagacac gggtatgtga tactctgtgt atatgtgagtctgtgtgaga 66480 gacactgata ctccaagaca ttgtgtgtgt gtgacactgt gagacaccaagacagtatat 66540 gtgcgagaca gcctatgtat gtgacagtgt gtgtgtgaga caccatgagagactgtgtat 66600 ttactgtgag agactgtgtg agagacacgt gtgagacact gtgtatatgacactatgtat 66660 acgtgagact gtgagacact atgtgtatgt gacactgtat atgtgtgagaccgtatgaga 66720 cagtgtatat gacactgtat atgtgtgaga ctgtgtgtgt gaaacactatatgacactgt 66780 atatgtgtga cactgtatat gacactatat gtggagacta tgtgtgagacactatgtata 66840 tgtgacacta tgtatgtgtg tgacactgtg agagacactg tgagacaccaagacagtata 66900 tgtatgagac accctgtgtg tgtgacacag cgtgtgactg tgtgagacgtgtgtgaggca 66960 ctgagactga cactgtgtgt gaggctgtgt gagagtcagt gtgtgtgataactgtgtgtg 67020 tatcaccgtg tgtgtgtgtg agagagggaa ggagagagag ggaggcaggtcagagggagt 67080 caggcgtcct tggcgagagt ggcagcagcc tgcctggagg gaccctgggatggccatttc 67140 agcacctaag gggtagcctg cccgggtgag tctccagtcc actgtgactcagtcattgtg 67200 cctgtgatcc ccaccctcca tctgcttgct tcccccccat ttgtcttccccagccccctc 67260 cgcccctccc cagaaggtga tgtgtgtgag catgggctcc accacggtccgggtaagttg 67320 ggtcccgccg cctgccgaca gccgcaacgg cgttatcacc cagtactccgtggcctacga 67380 ggcggtggac ggcgaggacc gcgggcggca tgtggtggat ggcatcagccgtgagcactc 67440 cagctgggac ctggtgggcc tggagaagtg gacggagtac cgggtgtgggtgcgggcaca 67500 cacagacgtg ggccccggcc ccgagagcag cccggtgctg gtgcgcaccgatgaggacgg 67560 taggcagtgc caccggggcg ggaggggagg cgttctgcct cagacaccacccaccaagct 67620 ccccagggcc ttcctttcct gaacacaggc ccaggtcaac tcatctttctggttcaggtg 67680 taatggccta aagtgggggg atgtcactta cgggataact gaggcccaaatcccagcctt 67740 tggggctgtc tccaaagcag ctgaaacctt cacaggctaa gatgggagaagcagccctgt 67800 ctaagatttg aaaggtcaag attgggcaga ttggtgtaaa agatactcaaagtagaatca 67860 gcaagactcc acgttggctg gacattggca tgattggggc ctaggaggagtcaggacagg 67920 ttgtgctgac taggggtggt caaagacctg gtgccccacc tccacctgttcccccatgtc 67980 gaccctcccc accaagtgag aggcctgggc cagaggggtg ggcagggccagtcctgggct 68040 cttacccgtg gcgggcagag ggagccttcc gtgtgcctca ccagggacagatgatctaag 68100 gaaactgtat cagggccttt cctgggggag tggggtgctg aggcaggaccctcaagtttg 68160 ctgtgcccac ctgagctagg gttgatacct ccaggcctga cttccttctctacctgaccc 68220 cccagtgccc agcgggcctc cgcggaaggt ggaggtggag ccactgaactccactgctgt 68280 gcatgtctac tggaagctgc ctgtccccag caagcagcat ggccagatccgcggctacca 68340 ggtcacctac gtgcggctgg agaatggcga gccccgtgga ctccccatcatccaagacgt 68400 catgctagcc gaggcccagg tgcagcattg ggtggtggtg gggtggcagggtgagcacag 68460 accagcatgc acaagctccc ttttggggcc cagatatgtc cctcttcccctgcctgccct 68520 cagcagtgct gtgactgcct ttccttggtt gtgagacccg agatgctttgcagcatcagg 68580 ggttaggctg gggttttttg ggtgtgggtt ttttgtttgt ttgtttgttttgagatagag 68640 cttcactctt gtcgcctagg ctggagtgcc tcctgggttc aagcaattctccttcctcag 68700 cctcccgagt agctgggatt acaggcgtct gccactgcgc ctagccaattttcgtatttt 68760 tagtagagac agggtttcac catattggcc aggctggtct cgaactcctgacctcaggtg 68820 atccgcctgc ctcagtctcc cagagtgctg agaatacagg tgttagccactgtgccccac 68880 caggtcgggg ttttgagatg tgcctttccc ctagacagtg ctgggctggcatccactctt 68940 cccacagaaa gggtagagag agtgctccag ggctgtctta ccccactggcggccatgggt 69000 ccgtggttgc tgcaaagctc tgtgagtagc caagtagagt gttgccctgctcctggccct 69060 gcagggaacg attcagcccc tgatgttcac cctcaagagc taggcctgctggccagcctc 69120 acacctccct ctgtgcacat ctgtcttcct gggaggatgg tcctgcccttgagcttgggg 69180 tgaggtccct ggggtattct gaacactggt tgctattcag atgaagaacttggaatgctg 69240 gggggttatg agagtggtat ggaattattc agcaagtagg tggctgctgcgcttggatga 69300 gaagccatgt ctgtggaccc ctaggaaagg gccacagttg ctgtcatgagccccctccca 69360 aaagaccctg ctggagagtc acaacacctg gtgtggtgct ctcaaggtctccctatccag 69420 gagcagggcc tccccattga gcctctcacc tctgcctggg tggagagcaggggtgcgtgt 69480 accactcaat gctgtacaca ctgtgcagag gggtggggtc acccacacagacaggagcct 69540 tattcctcca gctgggctca gccatctgaa gcaaaattct ttctgcccagagggtgctct 69600 cttccccctc tcagcctgcc cagtgctaag gcatccgggg tggaggaggcaggcatgtgc 69660 acccacctgc attcctgggg caggttgagg gctccttgtg gagcctcagtgaacacacct 69720 acccagaaac atcccaggaa tgggttcccc tagcccctcc tctctgagggggccagtggc 69780 cacagctgtg gccagtgggg ttccagagaa gccacttcaa gtgcccccttctggtcccaa 69840 gaggttcgga agggagctgg gccagaggct cagggacgct gcccatttagtctcagacat 69900 cccatcatgg gggcggtaac tcgagtctgg gctcccggag gacactgaagatggaggcct 69960 gctccgtagc cctctcagga ctgggtcatt ctgttcctcg ccagtgggagagcagtgggg 70020 cctggtcccg agcgtggcca cagggcccag ctgtccctgt gcttctgtcaagggcggcac 70080 attctgtcct tcacgcaaca gcaccatccc ctcagctcat cccccatcatgcctgaagga 70140 actatgtggt gcaggttccc acccccagcc tggaagtgtg gagccagacctgtctccttt 70200 tacgtcagat tccatctccc tgcctccctt tttcccctct cgtggcctgcatttctctat 70260 ccttcttgga gtctctgttt ttgtcttgct ctttctcttc tctctcctccttccttccct 70320 cctccccctg ccattcctct gacccctttc cttcatctcc atttggtttcatcctccgtc 70380 cttcccttct ttcttgctct gcctctagcg caggacaggg tccatgtgatgtgagaacct 70440 ccacgccaag gcttggttgg gacagcccag gtctccccgc aggcaaggagactggaaggg 70500 acgtgggccc agccacaccc tataaagtgg ccatgcacta aggacctgtccagagtctcc 70560 tctcatattc tgttccatgc ttcttgcaag gactttccaa cagagtgtcccaggacagga 70620 ggaacttagg ccacccagca tggaggccag tggacagagg ccagaccggcactgtggggg 70680 tgcttgggct ggaggacccc aggtacttcc ggcttggaga catcctggaccatctccctg 70740 tgatgtttca tggggcctca gaatggagac ctcacagtcc ctccacctgtccatctagaa 70800 tatctacatg caccaaaggc ccgcaacact gccagccccg aaacattccttcctacctta 70860 atcctcagac accccgagga aggaacgggt ataggtgatt tcccgtgcctgtgggctaca 70920 gcccaggctc tgagcttggc actcacagcc ttcatagtct tgctgactggcttgcccttg 70980 ggcccaccct ggccatgtgc cctgttcacc cccaactctt actgttgtgggacacttctc 71040 atgcctgggg tgattgtgta atgtatgatc acatctggga tgctttggcgggtggaagga 71100 gacactgaat agtcaattcc atggcaacaa gcacaggcca ggactgtccgaggcaaacca 71160 gggcatatgg gcaccccatt cacccctcag tgtcttcctt tcccttggcctgagctgttg 71220 aacccaccct tcttgtctgg cagcctctcc ctcccagcaa gcccaaaattgtcagagcat 71280 ctgtagctgc tttgtgttct tagggccttc tgtcagcggg tcctctccctgcaggctgcc 71340 tgccctccct gtctccctac cctcctcagc cctggcacac ccagttggtgctcagtgagg 71400 cagggattca aatctttgaa ccgggagttg ttctggggtg ctacccccatgggtactttg 71460 aggcccaaaa gccctccctc tgtcctccct gggcaaggtc ccttccaggccaggccctct 71520 ccaggtcaga gtccttcacc atcctgtctc cctttctctc cctctcccgcggtcagtggc 71580 ggccagagga gtccgaggac tatgtaagta acaggtgtgc gaacgcggacaagacatggg 71640 tcaagctggg ctcgtgggac gctcctcctc tccctccttt cctgctagcctgcactgcca 71700 agatccacag ggctctagcc acatcaggag aaaattggcg tttagacacaagcactgagc 71760 tgagcagcga ttggcatttc ctctaatcct tacttcttgc ctgtcgagcagcaatttgag 71820 gccagtgttc atgatcgacc agggcctggc ccctgccccg gccagacagggatggagtta 71880 aatccaatcc tgatcgttag gccttattga tccctggagt gaaaaatcacctgctttagg 71940 gcccaggctg ggagggctgt ctggagagtc ggattctggc attggtgcattctggagccc 72000 cagccctggg agaccctcca gctgtggcag gaggggtcca tgagggggtggtggcagctg 72060 caggggcccc actcaaggcc agagctggag ggataccagg gttgatgacagctctgttcc 72120 cactgcacgg tggtccctgc cctgcttccc actcttctct ctgtgtggtgtggcttgacc 72180 tcccatggtg tttctccgca tgtccagaga tgatgccttt gctgcagatgggtatatggg 72240 cagggtctgc caagcgggga ggacattgcc ctggctgctg tctcaggcatcactgagaac 72300 agatgtggaa gccagttccc caggtgtgga ggttttctta ttctcctagcccctcccctg 72360 cctttctcaa gaggtattgc agggtataga cattcacaga gttagtggcctatatggggg 72420 cagcggagtc ctgactgggt cctataggat ggcaccttag cccatcctgccaccttgctc 72480 tgtctgtgca tgcatgcact ggtgtccaca ggcagcctta cgcctgcttatgcaggagct 72540 gtaggctgtg tgcgtgtggc tgccaagagc cacgcaaggt gctgggtgcgtgcgaggctg 72600 tgccctgttg atatcctcag tctccgttca cagcacagtg gagtgagggaaacagtctgg 72660 ccctttgttc ttgtctgaaa agaataatga gctgtctgcc ccaggcgtgcggctcctggg 72720 atgggcgggg tcctcccagg cctgcatcct acctgcctgc ttcctctccagcagaggcca 72780 ccattgtata gccccacctt ccacaacccc tggccttgtg tgccccggggctcccctcag 72840 gctagggtcc tgaggtccct gacaaggtct ggcctctccc tgcattcttgtgatgggaac 72900 gaacccctcc tcctccctca ggaaaccact atcagcggcc tgaccccggagaccacctac 72960 tccgttactg ttgctgccta taccaccaag ggggatggtg cccgcagcaagcccaaaatt 73020 gtcactacaa caggtgcagg tgagtgaggg gtcaggacgg acctgagggtggggcagcag 73080 gagggcagcg ccagagccca gcccgtggtc cttcagtccc aggccggcccaccatgatga 73140 tcagcaccac ggccatgaac actgcgctgc tccagtggca cccacccaaggaactgcctg 73200 gcgagctgct gggctaccgg ctgcagtact gccgggccga cgaggcgcggcccaacacca 73260 tagatttcgg caaggatgac cagcacttca cagtcaccgg cctgcacaaggggaccacct 73320 acatcttccg gcttgctgcc aagaaccggg ctggcttggg tgaggagttcgagaaggaga 73380 tcaggacccc cgaggacctg cccagcggct tcccccaaaa cctgcatgtgacaggactga 73440 ccacgtctac cacagaactg gcctgggacc cgccagtgct ggcggagaggaacgggcgca 73500 tcatcagcta caccgtggtg ttccgagaca tcaacagcca acaggagctgcagaacatca 73560 cgacagacac ccgctttacc cttactggcc tcaagccaga caccacttacgacatcaagg 73620 tccgcgcatg gaccagcaaa ggctctggcc cactcagccc cagcatccagtcccggacca 73680 tgccggtgga gcaaggtgtg tgctgtggac atggcatccc ttcccgagtgtggctgcatc 73740 tgggggtctc tgctctcctt gagccactga cctctggcga ctgtgatccaccagcctctg 73800 gtgtgtgacc tccaatctct catgactgtg accactaacc tctagtgaatgggcaccaca 73860 ttcttggtgc ctgacctctg ctgtccttaa cctactgacc tctgctgtatgaccttctga 73920 tctcttgtga ccttgaccca ctgatctctt ttgactgtgt cactattcttgggtgtgcaa 73980 cctcctgatc tttggtgtgt gacactaatc tcttggggcc atgacccaccgacctctagt 74040 gaacatgctc caccacgctc tggtgtgtgg ccacatgctt ctcatgaccgaaacccactg 74100 accctctgat cactctggcc tggtgtccat cggctcaagc ttttacactcgcgtttctgg 74160 agattctgac cctggttgct gtggcatccc ccgccctgtt tggtgctcactgtggaagag 74220 cttgggctgg gagttcaact gtgccgtttg aagctggctg ggagtggggcgcttggtact 74280 ctgcagccat ccttcaaccc cctgttcccc aaccagtgtc tcatcctggccattcacctt 74340 ccacccttcc agcccattca ccccacctca tatacccagc agagctgactctctctatgc 74400 ctttgcagtg tttgccaaga acttccgggt ggcggctgca atgaagacgtctgtgctgct 74460 cagctgggag gttcccgact cctataagtc agctgtgccc tttaaggtgagtaagggcca 74520 cggccagctg agcctggcac acacacaggc ctgctgggtg ctgtctttccagtcctaacc 74580 catgtgcatc cggctgtgga gcaggaatgt ggttgtgtat ccgtgcactgtgccttgcag 74640 cccgtggtag ggaacctcac ccaaaggcat tgattgcccc tcccgtcccccacagattct 74700 gtacaatggg cagagtgtgg aggtggacgg gcactcgatg cggaagctgatcgcagacct 74760 gcagcccaac acagagtact cgtttgtgct gatgaaccgt ggcagcagcgcagggggcct 74820 gcagcacctg gtgtccatcc gcacagcccc cgacctcctg cctcacaagccgctgcctgc 74880 ctctgcctac atagaggacg gccgcttcga tctctccatg ccccatgtgcaagacccctc 74940 gcttgtcagg tgtgcacacg aggtatcggg ggaggcgggg cagggctggaggtaaccagc 75000 agtgacagtc ctgattcctg ccctgcccac ccaggtggtt ctacattgttgtggtaccca 75060 ttgaccgtgt gggcgggagc atgctgacgc caaggtggag cacacccgaggaactggagc 75120 tggacgaggt acctggggag gggatgggga cactgacagc cccattgcagtggtcagctg 75180 tggccttcct gccctgagca ctgtcccagt gactctcaga ttcactccccaaattgaaat 75240 ctctcttctg gctggcagcc cgcccctctc tggagagagg gactctgaggaaaccatctg 75300 ggagtattca cagaactccc ggagggctta cgaagaatcc ctggggtgggatgtcgtgga 75360 gatgcctctg caggtctaga gaatgccaag ccctgtagac actgcagagcctcgcagatc 75420 tagaagctat ggagggctta gtgagcctta tagccaggag tccctgaatgtttgaaatcc 75480 ttcagtcctt tctcatagcc agtgtggcaa cagcctggtt agggtgggtcagcttacaca 75540 cagggctgct tctcatgggc tgatggggag gagtggcttc acggtgtctgttactctgta 75600 ggggcagtgg gttgggcagg tgtgggctct tacacggaag gtgagccttgatctcggccc 75660 agggagctga catcccaggc cacagcccca gggctggccg gcatgctccaaggcccctca 75720 tgacccccat gctctgctct gccagcttct agaagccatc gagcaaggcggagaggagca 75780 gcggcggcgg cggcggcagg cagaacgtct gaagccatat gtggctgctcaactggatgt 75840 gctcccggag acctttacct tgggggacaa gaagaactac cggggcttctacaaccggcc 75900 cctgtctccg gacttgagct accagtgctt tgtgcttgcc tccttgaaggaacccatgga 75960 ccaggtctgc ctgagccggc ttggctgtca gcaccctgat tccctgggcctggcctgaga 76020 cgatgccagt ctcaaacacc acaagacccc aggtctttat cagtttgggggcttcgagat 76080 cctggggcag cactaaagac ccaagatctg tcccggggat cctaagacgcggccctggga 76140 cccagaggcc agacctaatg tggctccagg gacccagtcc tgccaggtccaccttgtagg 76200 gtctgggaga ccaggcccag ggtaacccag acccagagcc ctttctccaggattgatagg 76260 cagagggtgg ggggttctca cgctgagctc acagcctgct gttctccaccgggccacaga 76320 agcgctatgc ctccagcccc tactcggatg agatcgtggt ccaggtgacaccagcccagc 76380 agcaggagga gccggagatg ctgtgggtga cgggtcccgt gctggcagtcatcctcatca 76440 tcctcattgt catcgccatc ctcttgttca aaaggtgagc actgccctcagagctccggg 76500 aacggccacc tgcccctcgc ctttcaggcc ctctccgggt gtggtgcctgtggagagcgt 76560 gcagccttgc atcctggacc ctgagcctca ggctcagcag gaaggcaggaagggcaggag 76620 cagtgtgtgt gcctacctgt gtctgcaggc ctgcatctgt caggtgagggagcctctgca 76680 gcagcctggg cgtgaggaac taaagcctca gagggttgtc tcatgtgcatccctggcacg 76740 tctgtctgtc tctgtgtttc tgtggataag agtgcgtctg tctcctcttcagttttaaga 76800 caccgccatc tcctgagctc ctacctcgag tcctttcctt ctgccaccattcctggatga 76860 ttccccagcc ctactctcct gcttaggcct agagaggtcg agctggaggcccacaggcca 76920 tggatgctca ctaacagaca tgactgatgg cctgctgtgc ccgtgccatccgcagcggcc 76980 tcagcctcag tgtgtctttc ccactcagtt ctctatcggc agtggccaccacctgcccta 77040 ggagtctccc ttcctatccg tgcaccttcc agcctgaaag ggatttccaaagatttaact 77100 ttgtcactct cagtttaaaa ccttttgatg gctgcccacg taaagcccaccctctttagc 77160 ctgacagcca aacacctggc taatctgacc cctgccacac tcctgacattgcgacactga 77220 actcattcag tgttcaggcc cacacagggt gtgccctctg cggatctccctccccctgcc 77280 cacttggcaa cagcaatctc atcgttcagg actcaagtcc atgctctggtgcctgcaagg 77340 ccttgcctgg cccctacccc ttccttgcac agctgtcccc tgtgatgctccactgcaaac 77400 gcagctgggc ctgtgtgcca agccttcagc ccccagcact gggctgagcccacaggcatg 77460 tcagtgagca cctggcagat gaagatatgc tagtctgtgt gtctgagcacatctctgcac 77520 catgcaggag agcctgtatc ctgtgtgcct accctggacc cactgttccttcctaaagag 77580 tccactcctt cctcccaggg tggatggacc tgtgggcggg tccctaaggagtgtcccaca 77640 gatgcactca tccttcccac ttccacctgc attctgggtc tgtccagagaggtagacccc 77700 ctccctgtga atgtcatgtg ggtgccatat aaggggcctg cagggtccccatgtcattag 77760 agggctgagt gcttggtgga aactgtgacc gttcctttct tctgtccttgcttgtctttc 77820 gggacctagt aaacaagaaa ggtaggagtt gttccttctc tcttcacacacctccccctc 77880 tacttctaat ccttgggcca gcagggaggg atgacgcttt gaccagaagtcccctggcct 77940 atggctttaa gcagcaaact gtccagtcag ctcagcaggc tccctggttcaggactgagt 78000 cctcattggt gtgaggaggc agtttctgtc tcactcttgc cagttcaactctgcagagga 78060 tgcttcttct ctggggcaca tacataatac agactgcatt gctttccctgagcgtacaga 78120 gctcaccaga accatcaccc ggctcccagt tctctgagaa gttgcctaagtgctgcgaga 78180 gacagtctcg cacagagcaa agagcagtca ctgtgtggaa cagcagagaggcatgtcctt 78240 gtacccacct gagcacactc actcaaattc ttcttcccga agcacatgcgtctgcagacc 78300 acaagtagcc tctggctatt tgttggcagc tgaatcctat ggtctgtctctgaccctgtc 78360 cttgtagatg ggaaaactga ggccagtgag agaggactct gcccagctagggtgacgcct 78420 ttcccgtccc tcccctgctg ccctgggcat ttaccctgat actctgagggccaaaaagct 78480 ggggccctgt ctcctgtgct gtttgtgtat tttatgtgtc atttgcatgtttgttcccgg 78540 tggttcctgc tactcagctg ggtggccttg gagatagcag tcactgtcctgcagtgacct 78600 gccttctatc caaaggagct gctcgcccct gcaacccctt cctccctgctgcactgcccc 78660 tctgctcacg ccacactcct gctctcccta ccccctccct gccctgtgttctctcatgtc 78720 aaggctgttc tggccctggc tcctccgcgc tcagagatcc tgggaagaggtggggcagta 78780 ggaggacaga gccgtggaca tgggggctcc ttggcagcca gccatgccatgtgtcactgt 78840 ctcagccggg acctcaggtt ctcaccagcc tcccttctgt ctctctaggaaaaggaccca 78900 ctctccgtcc tctaaggatg agcagtcgat cggactgaag gactccttgctggcccactc 78960 ctctgaccct gtggagatgc ggaggctcaa ctaccagacc ccaggtagggcactcctatg 79020 gcctgtgtgc ccccagccca gacctagcag gcctgtgggt ccgttctgcaggtctcaggg 79080 tcgccactga agttcctggg ccgcatgcac ttgttcctgc tccttttcttcctttctgcc 79140 cttctccctg tgctcatcca ctttggttgg aatgactggg gagcccccatatcctgcagc 79200 tggacaggtg ccccaggcca ggaccctctt aggtggcaaa gccacttagccactgccttg 79260 tcttcagtca aggcctctga atatgggctg aggacctccc agagcccacgtctctgccac 79320 agggtggcca gtgtctggcc cacgctctga cgcctccaca tgaaagaggggcatgttagc 79380 agtgggagat gcctcaccag acatctgtgg ggagatgaag tgcctgcacctgagccttcc 79440 tggaggccga ccccaggttt accataccac gtacctgccc cttctcctagccactctcat 79500 ggggctgtcc acctcttcct tctacctgaa atgttactcc tcccaacacccagcagacac 79560 agctcagacc tctcctcttg cccacacccc cacagggtta ggtgtctcttcttggctcca 79620 ggaacctccc tccttcttcc ttttgacgcc tctcacagtg agctgggattacctcagtga 79680 cagtctctga gctctgtggt gccaggaact gtgtcttgtc atcccccatcccttgtattc 79740 agcgcacgat gggttcttgt tgaatgcctg tgaaatgaac acacagacaaatgaaggaat 79800 gagtgaacta atgcatacac acagggtttg tgtatgtatg agctggttattctgcatgtc 79860 aaattacccc aaaacttagt ggcttaaaac aacaataatt atgtattatctgtcacagtt 79920 tttgtgaggc aggaattcag acaggggcac agcaggaaca gcttgtctttattccagaaa 79980 gtctgggccc tcagccggaa gacttgaagg ctgggagcta gagtcatttaaagcaggggt 80040 tggcagacta tggcccgtgg ccaagtacag cccaccacct gtttttgtaaataaattttt 80100 actggctggg cgtggtggct catgcctgtg atcccagcac tttgggaggctgagccgggt 80160 ggatcacctg aggtcaggag ttcgagacca gctaatgaaa tcccatctctactaaaagta 80220 caaaaattag ccaggtgtgt ggcccctgtc atcccagcta ctcaggaggctgaggcagga 80280 gaatcacttg aacccaggag gtaaaggttg cgtgagccaa gatcgtaccatcgcattcca 80340 gcctgggtga caagagcaaa accccatctc aaaaataaac aaataaataaaattctactg 80400 gaacagacac acccatttat ttatatgcta cctacctatg gttgcttttgcactgtaaag 80460 gcaccattaa atagatgcag tagataccag atgtccacaa agcctaaactatttacgctc 80520 tggcctgtta tggataccaa gtttgccaac ccctggactc taaagcctctttcatccatg 80580 tgactggtgg ttgatgagcc agctctgagc cttgctgtgg ctgtcagctgggacacccaa 80640 catgaggctt cttcaggtgg cctgggcttc ctcacaacat ggtgactgagtttcatgagc 80700 atgtgtctgg agagtgtgcc aggcagacct agcaggctgt tgcaacccagcatgattaga 80760 tctctgaaga atcaagcacc aggatgcgat gctgggacct ggagtgcagagaagcctctt 80820 cggggatggg atgctgagcc atgtggttat tggtctggaa aagaaggatgggagagagct 80880 cctcagtgga agcatgtgca gaagcttggg cagattgcat gtgcgtcaaattatgagcac 80940 ctgagggttt ctgcagcatg agagttaggg gcttggggaa tactgagccaagagaagact 81000 ggagggctag gcagaggtcc agtcaaaagg accttgaatg tcaggctaaggggcttagac 81060 ttcctcttca ggatgccagg gaggtaggta ttaaaggttt tcagcgggggagccaggtgg 81120 tcagaaccat gctttagaaa tgtcactctg gctccttgag gagactggattagagaggag 81180 aaattgaggc agggagacca tttaaaaggc cagcaaccaa gtgaaaggtggcagtgggaa 81240 cagaggggac agacttggga aacgtgaagg aggtgaggac agcagagtccgggcttaagc 81300 ccacttgggg agaggtaagg gtggctctgc agttgctggc tgaatgagcagggggtaggg 81360 gtgtcctggg gctggtgggt ctgtgggtct gaggacttct gagctgacatcagggctgaa 81420 gccctgtggg aggggccaag gagcatgtgg tagtcaaagc ctgagaatagatgggctctc 81480 ccagggtgag ggccagaagg agaccgggac caaaaagccc agggcagggaaggtggtgca 81540 gcattaagga aagatggatg gagagcagct agggagattg agaaggagcagccacagcca 81600 gacgagagca ggagcgcagt gtccctgatg ccagggttgg gggagttgtgcaagggacga 81660 gaggacttaa tgaggtaggg cagtggggac acccagtggg aggaccgttggctttggcag 81720 atggggcctt gaagagaggg tggtggccac gtcctgtggg cagaagtgttgcagtgagca 81780 gcgtggcaag tgggggctga gaaagtggag aaaccccttt caaagatcctgggaagatac 81840 ctgggaagga ggcaatgagc agggcacagt gggagagtag agggtgtgggatctgagaag 81900 gagggctctt ttttccaatg tggaaatcat ctgaaaatcg ccaagggcaaattttggtat 81960 caaaaggggc agggctggtt tggacttaag tatttagtca tagagccccccaaggctgcc 82020 gagccaccag gctttagaag tgctcgcctc tgggggtggg acacccagtctgtattaact 82080 gggagacaga aggagccttg acggaacttg tccgcagccc cagcccctcaccgccccctc 82140 ctcttctccg ccagcccctc gcaagcccgc tcggcacctc cactgtgtgtgatggtgctg 82200 taagcagaaa aagttaacgg gctctctttt cttgccccgt tgtttttttcgtttgtttgt 82260 ttgttttttt ctctgcaggt tccagtgtcc ccagttgccc gaatacctcaagttagtttt 82320 caaagttccc gtgtttgggg gatactttgg cttctgtgtc tgtttccactccttactttt 82380 gtttacccca tcgcctccat ccttccttga attcttctct ccccttccctttcttctccc 82440 ccaatcccac tgtctcctaa cctttttcct tccctaccct cccctctgcccaccttgctt 82500 cctccaggcc tgttttctct cccaccggcc ccgtctctgt tctgcctctctggcctccag 82560 tcccggcctg acacccttcc ttctgcgtgc ctctacctct catctcctctcctctgtctc 82620 acacccccct cctggtctct gcttctctct ctattgtgtc tgtacttcatgaccactcca 82680 tctacacact tggtgcccgg gatgacgatg gctgtgagtc tcccatggtgactgccaccg 82740 gtgaggggca ggagggctgt ctgcaggcag atgcgatgga gcccagctcctgtcacgtct 82800 gctgccaccg acctgggcgt cccacccctc ctgggaggaa ggaagcctctcttccatctt 82860 gagagacctg ccaggcaggg cctagtgccc ccactcagca ccccgccaccaaaacaggct 82920 ccacatgctc atggcacaac accgccctct gtcctctccc accctccgccatccctgtcg 82980 ccgcatgtgc tgctgtctcc atgccaccag ttccaagtgc tccatggtcacacatgttca 83040 catgtgcaca tacatgcgtt ggggctttct ctgccacact gctcaagcctcacactaatg 83100 ctgcctgtgt atgccctacc tcccctaggt atgcgagacc acccacccatccccatcacc 83160 gacctggcgg acaacatcga gcgcctcaaa gccaacgatg gcctcaagttctcccaggag 83220 tatgaggtga gatgttcccg ccccctacca cgtgcctggc ccaggcctacccaaaccagc 83280 tcctgtcctg tcctaggtcc cagctgtggt gggtgaggaa gcaggggtccagctttttca 83340 ggagcacaga gaggagggtt ggcagtggta agggtcagct gggaaccgggtgcctcagat 83400 gctgggtctg gccatagcct ggcccagcac cttcttgggg tcacccttaggagatggttt 83460 caaaaggctg tgagtgacac cagggtctgg acactcagac acgtgctcaagtgctcacag 83520 gcagacacaa ggccacaggc atacagacat agatcagtga taacagccacagtagctggc 83580 atctatcaag agcatcctgc aggccagcac cggtctgggc acttggcatgcattatctct 83640 tttggttccc ccacagtctt cagaaatggg gactgttatt atcccatcttccaggtgagg 83700 aagcgggggc tcagagaggg gaagtgactt gcccaagtca aacagctgatgagtagtgga 83760 gccaggattc aaacccaggc ctgcctgctg ccctgtgctc tgcacatgcatgtgctcacg 83820 tgtgtactcc gatgccacag ctcacgggga gtcggggcct cgagactggctgctcaggct 83880 gtacaagtcc cgcttggagc cctccacacg ttcatcttgt tctggacttaactcctagga 83940 gcctgctggg ggctgagcct tcaggagtct gagggtttcc cacccacagatggtgtcggg 84000 gtgactctga gcatccccag gctgcccatc taggaagggg atttgttagagaaggaggtg 84060 atttaaagac aagactcctg gccaggcgca gtcgctcacg cctgtaatcccagcactttg 84120 ggaggccgag gcgggtggat cacctgaggt cgagagtttg agaccagcctggccaccgtg 84180 gcgaaactgc atctctacta taaatacaaa aattagccag ctgtggtggcacatgcctgt 84240 agtcccagct acttgtgagg gctgaggcag gagaatcgtt tgaacccgggaggcggaggt 84300 tgcgttgagc caaggtcatg ccattgcgct ccagcctggg tgacagagtgagactccctc 84360 tcaaaaaata taaaataaaa aaatactctt aaaaaagagt atttttacttaaaaaagaga 84420 gagagagacc tcctcctctt ccacctcctc caagcagcag cctgtgcttgtcgttctgcc 84480 ttgtccacag ctgtttcctc agcacctggc actggcctca gtagatggttggtggacagg 84540 caattgaggg gttggctgag cctaacctgt gagtttgcgc cccttctgatgtccaccctc 84600 agctgtgttt gggggatgca tcctagggct caaagattgc ctttcccaagggctgtgggc 84660 caggtttctg aagagaaagc tgggcttggc aggcaaatgg atgagtatgtctgcggcaca 84720 gcaaccgtgc tgcctctgcc tatagggccc ccctggggcc ctgctccacacaaggctggg 84780 ctttgggtca cggagcccgt aaggtgggct ccctgcctcc catggcctccacccacactc 84840 atctgtacca tgtcctacac ctgcccttcc ttccagcgca gccctgcttccccatctggg 84900 ctcgtggggc ctctgttcac agtagtcccc ttccatcttc tacctgcttccctcctctga 84960 tcagagcttt ccttacaaga ccctcctcct ccaggaagcc ttctcaggctccccaaggct 85020 gccgtgggct ctccctcagc caggacccca tagctctggt gtcctttttttttttttttt 85080 tttttttttt tttctttttt ggaagtctcg ctccgtcatc caggctggagtgcagtggcg 85140 tgatctcggc tcagtgcaag ctctgcctcc tgggttcacg ccattctcctgcctcagcct 85200 cccgagtagc tgggactaca ggtgcccgcc accacacccg gctaattttttttattttta 85260 gtagagatgg ggtttcaccg tgttagccag gatggtctcg atctcctgacctcgtgatcc 85320 gcccaccttg gcctcccaag tgctgggatt acaggcgtga gccactgcaccaggcctgct 85380 ctgttgtctt taacgttcag tcagtcattg atcacacatt ttccatgtgccagtccccgt 85440 gctggggatg tggagacaca tgccctcagg gaacccagtc gtggggagcatgtgcagaca 85500 gataatcagc attgaatatg tgactctgac agcatgccct ggtgttgaataggaccgaca 85560 ggcgtgcaaa aggtgggctg acagcaccag agcagcctga ggggtggggtgtgctgcggg 85620 gtctcacgga agcagtgacg cggtcctgtg gagagtaatg aggctggggacaacatatgg 85680 aggacattgc gtgctatgct gagaacttca gatcgtatcc atctggaaaatgtgaagata 85740 ataatggaac ctatctcgta tgagaatttg aggagggaat acatgcaaagtgctcacagc 85800 ggtgccaggc acactgtggg tcctcactaa cgcttcgcta ttatcattattgctctccga 85860 ggtgaagaag agccacagaa ggcctttaag caggaaattg atattcacatctgcattttg 85920 gaaggattat tctggctgtg gggctgagtg ggtcagagtg ggacaagaggaaagaggcag 85980 gaagaggctg agcctcaatt ggggtggcct ggcccagggc agtggcagcaggttggagag 86040 agctgagggg gtggggaagc accagaacct ggtgactggc agggcggggttggtggaggc 86100 cggaggaatt ttgagcttga cttctaggtt ctggcctggc actgaggggtgagggtgcct 86160 ttcgctaagg tgtagtccca gcagcagcgc caggtttgga ggggaagctgatgaactcag 86220 ctttgaaagg actgcggttg aaggacctcc cggtggaggt gtcagagaggctgctgggta 86280 ctcggggaag agctctgtgc tgctgctgag gttatgggca gctccagcatgtggtgtaag 86340 aatcaggtga gcaaatgagg tcacccagga aaggttgtag agagagaagagggcctagaa 86400 ggaaccctgg aggagctctg cgtcctgccc acgggaaggg caggggagagagaggaaaca 86460 gagaaggagc agctgaaagg aagagagccg agcagggagg gatcctggactacctgagag 86520 gggccaccat cccttccatc tgtcctggct catttcatta ggtgaatgcagggcttcggg 86580 agggactctg ggtgttcaga gccctcagca gtttcgggac tgcctgagagggggccacca 86640 cctgtcctag ctcatttcat taggtgagtg cagggcttcg agagacccttcacctgcccc 86700 atcccagctc agaccactct accaggcaag gggatttggt accatggtcagaggagtccc 86760 cagtccatca cttttctgat aggtgatgtg gcaacctgtg agctccatggctggcaccac 86820 gagatagagg gcctggctgt ggcctgtgga gtgaaagcag gatgtgagcatcaccgggaa 86880 ggctgggtcc cctgcaggag aagcaggtca accttggctc ttaccccaccccacccgctt 86940 tctccattct ttgcagtcca tcgaccctgg acagcagttc acgtgggagaattcaaacct 87000 ggaggtgaac aagcccaaga accgctatgc gaatgtcatc gcctacgaccactctcgagt 87060 catccttacc tctatcgatg gtgagccaag ggggtgcccc tcccatccccttgctctccc 87120 ccttgctagc tagggcaaca tgtcattcta cagaggatgt ccacgagtctcaggggtgca 87180 ctgaggcatg gtgggctggg ctggggaccc tgtagtaatg ccctcccacctcctttctta 87240 tccataggcg tccccgggag tgactacatc aatgccaact acatcgatggctaccgcaag 87300 cagaatgcct acatcgccac gcagggcccc ctgcccgaga ccatgggcgatttctggaga 87360 atggtgtggg aacagcgcac ggccactgtg gtcatgatga cacggctggaggagaagtcc 87420 cgggtgaggc tgcagggccc tgccaggagg cgggtgggaa atgcccagccacaaggtgat 87480 acagggcacc ttcttctgtg ccgctttctt ctgtggagga agtcgctcaagtgatcccca 87540 gatgctattg ttactggggg tattatgctc cccaaatact gggtgtttctgggatacagc 87600 atgttcccca catgctagtg ggttccttaa gatgttaata tgtttcaccaaatgctgtca 87660 ttttcggaga atgttaatgt gttccccagt tgctggcgtg ttcccggggtattcgtgtgt 87720 tccccagatg ctggagtgtt cctggggcat taacacatct cctaagttaattagtggaaa 87780 gagctgctgt tatctgtttt tggactgcaa agcattgact ggaacgtaaaatttacagag 87840 cttgaaaatg gcacataaac gttatgggta aatttggcag aaaatgtgcctggtgcgatc 87900 tggcgttgaa taaataattt tcaattatga cccttattcc actagctagggcagggcggt 87960 gacatagctt gaggacctga tgtggcttgt ggaggacagg gggcaatggatctgagagct 88020 cagggctggg gggctttgta gccagaaagg ctacagccag ggagttgaccagcctccacc 88080 ctgcttctgc ccgtctgagc ctgtgggctt ccttcagcct gccctgctcatcctcctgca 88140 ggtaaaatgt gatcagtact ggccagcccg tggcaccgag acctgtggccttattcaggt 88200 gaccctgttg gacacagtgg agctggccac atacactgtg cgcaccttcgcactccacaa 88260 ggtatagcct ttccccagtg catatctctt acccagacac tgtaaggacagtggcctggg 88320 tgtggtgtgc tgggtcgggg ggaagctgga gcctgggtgt tggagggtcggaggctcagg 88380 tgtgtgagtg atgtgatgat ccatgttatg ggaacagtgc taggagcttcaggctactct 88440 gtgtggcttc tgagtcccat ggggaagtgg cgggtatggc ctcagcatcaggtcattcag 88500 tcctgagtct atggcaggta ggctcctagt cgccagtatg tccccactttgtcccccaga 88560 gtggctccag tgagaagcgc gagctgcgtc agtttcagtt catggcctggccagaccatg 88620 gagttcctga gtacccaact cccatcctgg ccttcctacg acgggtcaaggcctgcaacc 88680 ccctagacgc agggcccatg gtggtgcact gcaggtgaga gggtacagtgccacccagag 88740 gggtgggtgg ggtgggaggt gggggcgcct gtgcctcaag ctgagcccgtgtcctgcagc 88800 gcgggcgtgg gccgcaccgg ctgcttcatc gtgattgatg ccatgttggagcggatgaag 88860 cacgagaaga cggtggacat ctatggccac gtgacctgca tgcgatcacagaggaactac 88920 atggtgcaga cggaggacca gtacgtgttc atccatgagg cgctgctggaggctgccacg 88980 tgcggccaca cagaggtgcc tgcccgcaac ctgtatgccc acatccagaagctgggccaa 89040 gtgcctccag gggagagtgt gaccgccatg gagctcgagt tcaaggtggggctcgggtgg 89100 gcctgcttgg ctccagggcc tagactgggt catgcagatg acccccacccccacaggaag 89160 cctggcctga ccaatccctg cctctcaata gttgctggcc agctccaaggcccacacgtc 89220 ccgcttcatc agcgccaacc tgccctgcaa caagttcaag aaccggctggtgaacatcat 89280 gccctacgaa ttgacccgtg tgtgtctgca gcccatccgt ggtgtggagggctctgacta 89340 catcaatgcc agcttcctgg atggttatag gtcagcatgc atgtcactgccccaccatgc 89400 cctacagggg cctaggcctg tgcctggctg gtgggggtgg gcagcagagtagggccagcc 89460 tagaagacca gagagggctg ggtagagcag tgaggacttc ctggaggaggggtgatctga 89520 gcagggcccc aaggggctag gcagcctaag gggagactct aggggcagcagcacctccag 89580 catgtccagt cttatgtcca ccccagacag cagaaggcct acatagctacacaggggcct 89640 ctggcagaga gcaccgagga cttctggcgc atgctatggg agcacaattccaccatcatc 89700 gtcatgctga ccaagcttcg ggagatgggc agggtgagcc caccctttcccccagggccc 89760 ctgtcatacc tgggagaaca ccagccaccc ttgggggagc tgccgcctatgttactgtct 89820 cctttgacac cccagctgct tgtcagcatg gcctcaggcg cccgttattactacctgagg 89880 catctgtccc agaatcctgt gaagcctggc acccctcccc tattccttctcacctgatta 89940 tgggggcccg accctctgtc cacaggagaa atgccaccag tactggccagcagagcgctc 90000 tgctcgctac cagtactttg ttgttgaccc gatggctgag tacaacatgccccagtatat 90060 cctgcgtgag ttcaaggtca cggatgcccg ggtgagtgag tgcattgagtgtgtccataa 90120 cgctgcctgt ccacacgctg ggtggatggc tgcctgcatg gtaccttagctcaagcttca 90180 gaaatctgag gcggtgggtg ggtattaggg tgtgagcaca tctccccctgtggcctcggg 90240 tgcagtgaca cagatgcatg cctgtatcat ggtactaccc tggtctagtccagaggggtg 90300 gctgcctaag gcacgaattc taatcatgta ccccacccac ctttcccaggatgggcagtc 90360 aaggacaatc cggcagttcc agttcacaga ctggccagag cagggcgtgcccaagacagg 90420 cgagggattc attgacttca tcgggcaggt gcataagacc aaggagcagtttggacagga 90480 tgggcctatc acggtgcact gcaggtggga ctggccccct ggagggctggggtgggtggg 90540 cctgaaggcc tggcagaccc actgcatgag gcaagcagga ctcctgacccaactgtgttt 90600 ctgagcagtg ctggcgtggg ccgcaccggg gtgttcatca ctctgagcatcgtcctggag 90660 cgcatgcgct acgagggcgt ggtcgacatg tttcagaccg tgaagaccctgcgtacacag 90720 cgtcctgcca tggtgcagac agaggtaacg cagaccaggc tgcagggccagggccttggc 90780 agcagcgctg ctgggaaccc taggctttag caacagtttg atgcccacaggcatgtgcat 90840 tcattcatgc tgccaacctt tcacgtggcc tgcgataggc atggtggtgtgtgcttatgg 90900 tcctacctac ttgggaggtt gatgtgggag aatcacttga ggtcagaagttcgaggctgc 90960 agtgagctat gattatacca cggcactcca gcctgggtgg cagagcaagaccctgttgct 91020 gaaaacaaaa caaaacaaaa caaaaatgct gcaacattgc ctgtgctgcctgggggctca 91080 ggggattgat gagtaagatg tgttctttca ttcggcagct ctctgctgagccccagtggt 91140 gtgcctggct ctgggcaagg ttgcacagag caatccttat ggggtgcccagtcaggagca 91200 gagaaacatg attgggatgg cagatggaag gcaggtagat gtgggggcctgagagaatga 91260 caggagagag atgagccttc agaggctctt tcaggctccc ccgcacactgcctgatgtag 91320 ctgccagggt ccagcacctg ctcttggcca gcagaggcta actccatggctgcagtgtga 91380 gtgtcagctg tgtagtgggg gtgtccactg gcgcgaccca cactgaccagccccctatcc 91440 tggcaggacc agtatcagct gtgctaccgt gcggccctgg agtacctcggcagctttgac 91500 cactatgcaa cgtaactacc gctcccctct cctccgccac ccccgccgtggggctccgga 91560 ggggacccag ctcctctgag ccataccgac catcgtccag ccctcctacgcagatgctgt 91620 cactggcaga gcacagccca cggggatcac agcgtttcag gaacgttgccacaccaatca 91680 gagagcctag aacatccctg ggcaagtgga tggcccagca ggcaggcactgtggcccttc 91740 tgtccaccag acccacctgg agcccgcttc aagctctctg ttgcgctcccgcatttctca 91800 tgcttcttct catggggtgg ggttggggca aagcctcctt tttaatacattaagtggggt 91860 agactgaggg attttagcct cttccctctg atttttcctt tcgcgaatccgtatctgcag 91920 aatgggccac tgtaggggtt ggggtttatt ttgttttgtt tttttttttcttgagttcac 91980 tttggatcct tattttgtat gacttctgct gaaggacaga acattgccttcctcgtgcag 92040 agctggggct gccagcctga gcggaggctc ggccgtgggc cgggaggcagtgctgatccg 92100 gctgctcctc cagcccttca gacgagatcc tgtttcagct aaatgcagggaaactcaatg 92160 tttttttaag ttttgttttc cctttaaagc ctttttttag gccacattgacagtggtggg 92220 cggggagaag atagggaaca ctcatccctg gtcgtctatc ccagtgtgtgtttaacattc 92280 acagcccaga accacagatg tgtctgggag agcctggcaa ggcattcctcatcaccatcg 92340 tgtttgcaaa ggttaaaaca aaaacaaaaa accacaaaaa taaaaaacaaaaaaaacaaa 92400 aaacccaaga aaaaaaaaaa gagtcagccc ttggcttctg cttcaaaccctcaaganggg 92460 aagcaactcc gtgtgcctgg ggttcccgag ggagctgctg gctgacctgggcccacagag 92520 cctggctttg gtccccagca ttgcagtatg gtgtggtgtt tgtaggctgtggggtctggc 92580 tgtgtggcca aggtgaatag cacaggttag ggtgtgtgcc acaccccatgcacctcaggg 92640 ccaagcgggg gcgtggctgg cctttcaggt ccaggccagt gggcctggtagcacatgtct 92700 gtcctcagag caggggccag atgattttcc tccctggttt gcagctgttttcaaagcccc 92760 cgataatcgc tcttttccac tccaagatgc cctcataaac caatgtggcaagactactgg 92820 acttctatca atggtactct aatcagtcct tattatccca gcttgctgaggggcagggag 92880 agcgcctctt cctctgggca gcgctatcta gataggtaag tgggggcggggaagggtgca 92940 tagctgtttt agctgaggga cgtggtgccg acgtccccaa acctagctaggctaagtcaa 93000 gatcaacatt ccagggttgg taatgttgga tgatgaaaca ttcatttttaccttgtggat 93060 gctagtgctg tagagttcac tgttgtacac agtctgtttt ctatttgttaagaaaaacta 93120 cagcatcatt gcataattct tgatggtaat aaatttgaat aatcagatttcttacaaacc 93180 aggactctgt ctcagctgtt tctggaacca aagagtctgg gccaaatcagtagctaggat 93240 gggttctgga gattccctgc tcctgaggag acggggaggg taccctaagtatttgtgcca 93300 gtgtaggctc ctggcatggc tacccacttt cagaaaggag tggttataaaccctgaaaac 93360 caggccacca agagccagcc aggccagagc caccagtgca tgtgaagagcaccctaggcc 93420 ggggagcata ccctttgcct ctctttccct ttaagattct gtgggttgcactatcggggc 93480 attgggactg cccctctccc actaccttgg aggagaggga tggccctgtggcagtagaga 93540 ctgaatgtat ggaaattggt tagtgagatc tcctgtaatt attgcaatgtggataatgga 93600 cacaaaaaac agtgtgtcca tctggcccct ggacacacag ctgcatcacccactgagctg 93660 tgcagctgct ccactggtga gcagacaagt cctaccaggt tgtcaaattgtggaacttct 93720 agggatacag atgaaggaga cccagaacaa gctgcgaaga gaaatgcgtaagtcagcaac 93780 aaccacacca aggcagcatc tgacccaggg aaggcttcct ggaggaggtggcattcagag 93840 tgtttcgtag aatgagtagc agttagtttt tttttgttta tttttgagatggagtcccac 93900 tctgtcgcaa ggctggagtg cagtggcgtg atctcggctc actgcaacctctgccccccg 93960 ggttcaagca attcttctgc ctttaccctc ctgagtagct g 94001 2120 DNA Artificial Sequence Antisense Oligonucleotide 21 gactttcttccccttcttca 20 22 20 DNA Artificial Sequence Antisense Oligonucleotide 22ttctccacca ccttcagctg 20 23 20 DNA Artificial Sequence AntisenseOligonucleotide 23 tggagaaacg aggagccacg 20 24 20 DNA ArtificialSequence Antisense Oligonucleotide 24 cccggaccgt ggtggagccc 20 25 20 DNAArtificial Sequence Antisense Oligonucleotide 25 cacggagtac tgggtgataa20 26 20 DNA Artificial Sequence Antisense Oligonucleotide 26 ctgatgccatccaccacatg 20 27 20 DNA Artificial Sequence Antisense Oligonucleotide 27tgtgtgtgcc cgcacccaca 20 28 20 DNA Artificial Sequence AntisenseOligonucleotide 28 accagcaccg ggctgctctc 20 29 20 DNA ArtificialSequence Antisense Oligonucleotide 29 tgcacagcag tggagttcag 20 30 20 DNAArtificial Sequence Antisense Oligonucleotide 30 gcatggtccg ggactggatg20 31 20 DNA Artificial Sequence Antisense Oligonucleotide 31 aagggcacagctgacttata 20 32 20 DNA Artificial Sequence Antisense Oligonucleotide 32agcttccgca tcgagtgccc 20 33 20 DNA Artificial Sequence AntisenseOligonucleotide 33 gtgcggatgg acaccaggtg 20 34 20 DNA ArtificialSequence Antisense Oligonucleotide 34 acaatgtaga accacctgac 20 35 20 DNAArtificial Sequence Antisense Oligonucleotide 35 gcgcttctgg tccatgggtt20 36 20 DNA Artificial Sequence Antisense Oligonucleotide 36 gaggatggcgatgacaatga 20 37 20 DNA Artificial Sequence Antisense Oligonucleotide 37ggtggtctcg catacctggg 20 38 20 DNA Artificial Sequence AntisenseOligonucleotide 38 ccatcgttgg ctttgaggcg 20 39 20 DNA ArtificialSequence Antisense Oligonucleotide 39 tcgcccatgg tctcgggcag 20 40 20 DNAArtificial Sequence Antisense Oligonucleotide 40 accgtcttct cgtgcttcat20 41 20 DNA Artificial Sequence Antisense Oligonucleotide 41 gtcagcatgacgatgatggt 20 42 20 DNA Artificial Sequence Antisense Oligonucleotide 42tgtccttgac tgcccatccc 20 43 20 DNA Artificial Sequence AntisenseOligonucleotide 43 agcactgcag tgcaccgtga 20 44 20 DNA ArtificialSequence Antisense Oligonucleotide 44 atagcgcatg cgctccagga 20 45 20 DNAArtificial Sequence Antisense Oligonucleotide 45 gccgcacggt agcacagctg20 46 20 DNA Artificial Sequence Antisense Oligonucleotide 46 gctcagaggagctgggtccc 20 47 20 DNA Artificial Sequence Antisense Oligonucleotide 47aacagagagc ttgaagcggg 20 48 20 DNA Artificial Sequence AntisenseOligonucleotide 48 acctttgcaa acacgatggt 20 49 20 DNA ArtificialSequence Antisense Oligonucleotide 49 gcccccgctt ggccctgagg 20 50 20 DNAArtificial Sequence Antisense Oligonucleotide 50 ctaccaggcc cactggcctg20 51 20 DNA Artificial Sequence Antisense Oligonucleotide 51 tagtcttgccacattggttt 20 52 20 DNA Artificial Sequence Antisense Oligonucleotide 52ccacttacct atctagatag 20 53 20 DNA Artificial Sequence AntisenseOligonucleotide 53 atcttgactt agcctagcta 20 54 20 DNA ArtificialSequence Antisense Oligonucleotide 54 gtaaaaatga atgtttcatc 20 55 20 DNAArtificial Sequence Antisense Oligonucleotide 55 ctacagcact agcatccaca20 56 20 DNA Artificial Sequence Antisense Oligonucleotide 56 gtttttcttaacaaatagaa 20 57 20 DNA Artificial Sequence Antisense Oligonucleotide 57gggcaccatc gtcctccctg 20 58 20 DNA Artificial Sequence AntisenseOligonucleotide 58 tggcttcatc tcgctgcacc 20 59 20 DNA ArtificialSequence Antisense Oligonucleotide 59 cgttgcggcc aactggcatc 20 60 20 DNAArtificial Sequence Antisense Oligonucleotide 60 tttggaagag ctttcactgt20 61 20 DNA Artificial Sequence Antisense Oligonucleotide 61 ggttgggtcgaaggtgacct 20 62 20 DNA Artificial Sequence Antisense Oligonucleotide 62cccatatccg agcgtgcagc 20 63 20 DNA Artificial Sequence AntisenseOligonucleotide 63 tataggcagc aacagtaacg 20 64 20 DNA ArtificialSequence Antisense Oligonucleotide 64 gcgggtgtct gtcgtgatgt 20 65 20 DNAArtificial Sequence Antisense Oligonucleotide 65 cagagccttt gctggtccat20 66 20 DNA Artificial Sequence Antisense Oligonucleotide 66 tgtacagaatcttaaagggc 20 67 20 DNA Artificial Sequence Antisense Oligonucleotide 67ggttgtagaa gccccggtag 20 68 20 DNA Artificial Sequence AntisenseOligonucleotide 68 cactggtagc tcaagtccgg 20 69 20 DNA ArtificialSequence Antisense Oligonucleotide 69 ggtccttttc cttttgaaca 20 70 20 DNAArtificial Sequence Antisense Oligonucleotide 70 tggccgtgcg ctgttcccac20 71 20 DNA Artificial Sequence Antisense Oligonucleotide 71 gtcacctgaataaggccaca 20 72 20 DNA Artificial Sequence Antisense Oligonucleotide 72tcatccgctc caacatggca 20 73 20 DNA Artificial Sequence AntisenseOligonucleotide 73 atcgcatgca ggtcacgtgg 20 74 20 DNA ArtificialSequence Antisense Oligonucleotide 74 tctgtgatcg catgcaggtc 20 75 20 DNAArtificial Sequence Antisense Oligonucleotide 75 ccgtgatagg cccatcctgt20 76 20 DNA Artificial Sequence Antisense Oligonucleotide 76 agcggtagttacgttgcata 20 77 20 DNA Artificial Sequence Antisense Oligonucleotide 77caatgttctg tccttcagca 20 78 20 DNA Artificial Sequence AntisenseOligonucleotide 78 aagccaggct ctgtgggccc 20 79 20 DNA ArtificialSequence Antisense Oligonucleotide 79 agccacgccc tcatagcgca 20 80 20 DNAArtificial Sequence Antisense Oligonucleotide 80 ggtcactcac cgcctatcca20 81 20 DNA Artificial Sequence Antisense Oligonucleotide 81 aactcccgggtaactccctt 20 82 20 DNA Artificial Sequence Antisense Oligonucleotide 82agaggcccag agaggttaag 20 83 20 DNA Artificial Sequence AntisenseOligonucleotide 83 actcaatgac ctgtcagagg 20 84 20 DNA ArtificialSequence Antisense Oligonucleotide 84 ttgatcctcc caagagcccc 20 85 20 DNAArtificial Sequence Antisense Oligonucleotide 85 gcactgccta ccgtcctcat20 86 20 DNA Artificial Sequence Antisense Oligonucleotide 86 tccaggacgatgctcagagt 20 87 20 DNA Artificial Sequence Antisense Oligonucleotide 87aacatgtcga ccacgccctc 20 88 20 DNA Artificial Sequence AntisenseOligonucleotide 88 tctgcgttac ctctgtctgc 20 89 20 DNA ArtificialSequence Antisense Oligonucleotide 89 gatactggtc ctgccaggat 20 90 20 DNAArtificial Sequence Antisense Oligonucleotide 90 ctgtccttca gcagaagtca20 91 20 DNA Artificial Sequence Antisense Oligonucleotide 91 tgaaaggccagccacgcccc 20 92 20 DNA Artificial Sequence Antisense Oligonucleotide 92gctgggagct gactttcttc 20 93 20 DNA Artificial Sequence AntisenseOligonucleotide 93 gctgcactcg taatggctgg 20 94 20 DNA ArtificialSequence Antisense Oligonucleotide 94 acttacccgg accgtggtgg 20 95 20 DNAArtificial Sequence Antisense Oligonucleotide 95 acccaactta cccggaccgt20 96 20 DNA Artificial Sequence Antisense Oligonucleotide 96 tgctcacggctgatgccatc 20 97 20 DNA Artificial Sequence Antisense Oligonucleotide 97agacatgcac agcagtggag 20 98 20 DNA Artificial Sequence AntisenseOligonucleotide 98 cttggcaaac acttgctcca 20 99 20 DNA ArtificialSequence Antisense Oligonucleotide 99 tcccgcccac acggtcaatg 20 100 20DNA Artificial Sequence Antisense Oligonucleotide 100 gcggtagctcttcttgtccc 20 101 20 DNA Artificial Sequence Antisense Oligonucleotide101 ccaggaacac catcaatgga 20 102 20 DNA Artificial Sequence AntisenseOligonucleotide 102 ctccagaaat cgcccatggt 20 103 20 DNA ArtificialSequence Antisense Oligonucleotide 103 cacacttcac ccgggatttc 20 104 20DNA Artificial Sequence Antisense Oligonucleotide 104 tggagccactcttatggagg 20 105 20 DNA Artificial Sequence Antisense Oligonucleotide105 gtcacgtggc catagatgtc 20 106 20 DNA Artificial Sequence AntisenseOligonucleotide 106 cccgaagctt ggtcagcatg 20 107 20 DNA ArtificialSequence Antisense Oligonucleotide 107 ctgcccatct cccgaagctt 20 108 20DNA Artificial Sequence Antisense Oligonucleotide 108 cggattgtccttgactgccc 20 109 20 DNA Artificial Sequence Antisense Oligonucleotide109 tccagggccg cacggtagca 20 110 20 DNA Artificial Sequence AntisenseOligonucleotide 110 agcagtagtt acgttgcata 20 111 20 DNA ArtificialSequence Antisense Oligonucleotide 111 ggtatggctc agaggagctg 20 112 20DNA Artificial Sequence Antisense Oligonucleotide 112 ggctctctgactggtgtggc 20 113 20 DNA Artificial Sequence Antisense Oligonucleotide113 cacttggccc ggtggacgag 20 114 20 DNA Artificial Sequence AntisenseOligonucleotide 114 gagaagcatg agaacgcgga 20 115 20 DNA ArtificialSequence Antisense Oligonucleotide 115 tccttccgca gaagttgtac 20 116 20DNA Artificial Sequence Antisense Oligonucleotide 116 cacaaggaaggcgagttact 20 117 20 DNA Artificial Sequence Antisense Oligonucleotide117 agtcacgctg cctcccgggc 20 118 20 DNA Artificial Sequence AntisenseOligonucleotide 118 ggacagcagg agtcacgctg 20 119 20 DNA ArtificialSequence Antisense Oligonucleotide 119 ccccagacac gtctgtggtt 20 120 20DNA Artificial Sequence Antisense Oligonucleotide 120 cccgaggtggccagaaccca 20 121 20 DNA Artificial Sequence Antisense Oligonucleotide121 tcacctgtgc cattcatttc 20 122 20 DNA Artificial Sequence AntisenseOligonucleotide 122 cagacatgtg ctaccaggcc 20 123 20 DNA ArtificialSequence Antisense Oligonucleotide 123 ctgaggacag acatgtgcta 20 124 20DNA Artificial Sequence Antisense Oligonucleotide 124 agctgcaaaccaggagagaa 20 125 20 DNA Artificial Sequence Antisense Oligonucleotide125 aagtccagta gtcttgccac 20 126 20 DNA Artificial Sequence AntisenseOligonucleotide 126 tgcccagagg aagagaccct 20 127 20 DNA ArtificialSequence Antisense Oligonucleotide 127 aacagctatg cacccttccc 20 128 20DNA Artificial Sequence Antisense Oligonucleotide 128 gcatccacaaggtaaaaatg 20 129 20 DNA Artificial Sequence Antisense Oligonucleotide129 acagtgaact ctacagcact 20 130 20 DNA Artificial Sequence AntisenseOligonucleotide 130 catgatcgct gtagtttttc 20 131 20 DNA ArtificialSequence Antisense Oligonucleotide 131 agatacagag ctgagacaga 20 132 20DNA Artificial Sequence Antisense Oligonucleotide 132 ggctcacccccttgggagga 20 133 20 DNA H. sapiens 133 cgtggctcct cgtttctcca 20 134 20DNA H. sapiens 134 gggctccacc acggtccggg 20 135 20 DNA H. sapiens 135ttatcaccca gtactccgtg 20 136 20 DNA H. sapiens 136 tgtgggtgcg ggcacacaca20 137 20 DNA H. sapiens 137 ctgaactcca ctgctgtgca 20 138 20 DNA H.sapiens 138 catccagtcc cggaccatgc 20 139 20 DNA H. sapiens 139cacctggtgt ccatccgcac 20 140 20 DNA H. sapiens 140 aacccatgga ccagaagcgc20 141 20 DNA H. sapiens 141 tcattgtcat cgccatcctc 20 142 20 DNA H.sapiens 142 cccaggtatg cgagaccacc 20 143 20 DNA H. sapiens 143cgcctcaaag ccaacgatgg 20 144 20 DNA H. sapiens 144 ctgcccgaga ccatgggcga20 145 20 DNA H. sapiens 145 atgaagcacg agaagacggt 20 146 20 DNA H.sapiens 146 accatcatcg tcatgctgac 20 147 20 DNA H. sapiens 147gggatgggca gtcaaggaca 20 148 20 DNA H. sapiens 148 tcacggtgca ctgcagtgct20 149 20 DNA H. sapiens 149 tcctggagcg catgcgctat 20 150 20 DNA H.sapiens 150 cagctgtgct accgtgcggc 20 151 20 DNA H. sapiens 151gggacccagc tcctctgagc 20 152 20 DNA H. sapiens 152 cccgcttcaa gctctctgtt20 153 20 DNA H. sapiens 153 accatcgtgt ttgcaaaggt 20 154 20 DNA H.sapiens 154 cctcagggcc aagcgggggc 20 155 20 DNA H. sapiens 155aaaccaatgt ggcaagacta 20 156 20 DNA H. sapiens 156 ctatctagat aggtaagtgg20 157 20 DNA H. sapiens 157 tagctaggct aagtcaagat 20 158 20 DNA H.sapiens 158 gatgaaacat tcatttttac 20 159 20 DNA H. sapiens 159tgtggatgct agtgctgtag 20 160 20 DNA H. sapiens 160 cagggaggac gatggtgccc20 161 20 DNA H. sapiens 161 ggtgcagcga gatgaagcca 20 162 20 DNA H.sapiens 162 gatgccagtt ggccgcaacg 20 163 20 DNA H. sapiens 163aggtcacctt cgacccaacc 20 164 20 DNA H. sapiens 164 cgttactgtt gctgcctata20 165 20 DNA H. sapiens 165 acatcacgac agacacccgc 20 166 20 DNA H.sapiens 166 atggaccagc aaaggctctg 20 167 20 DNA H. sapiens 167ctaccggggc ttctacaacc 20 168 20 DNA H. sapiens 168 ccggacttga gctaccagtg20 169 20 DNA H. sapiens 169 tgttcaaaag gaaaaggacc 20 170 20 DNA H.sapiens 170 gtgggaacag cgcacggcca 20 171 20 DNA H. sapiens 171tgtggcctta ttcaggtgac 20 172 20 DNA H. sapiens 172 tgccatgttg gagcggatga20 173 20 DNA H. sapiens 173 ccacgtgacc tgcatgcgat 20 174 20 DNA H.sapiens 174 gacctgcatg cgatcacaga 20 175 20 DNA H. sapiens 175acaggatggg cctatcacgg 20 176 20 DNA H. sapiens 176 tatgcaacgt aactaccgct20 177 20 DNA H. sapiens 177 tgctgaagga cagaacattg 20 178 20 DNA H.sapiens 178 gggcccacag agcctggctt 20 179 20 DNA H. sapiens 179tgcgctatga gggcgtggct 20 180 20 DNA H. sapiens 180 aagggagtta cccgggagtt20 181 20 DNA H. sapiens 181 atgaggacgg taggcagtgc 20 182 20 DNA H.sapiens 182 actctgagca tcgtcctgga 20 183 20 DNA H. sapiens 183tgacttctgc tgaaggacag 20 184 20 DNA H. sapiens 184 ggggcgtggc tggcctttca20 185 20 DNA M. musculus 185 acggtccggg taagttgggt 20 186 20 DNA M.musculus 186 tggagcaagt gtttgccaag 20 187 20 DNA M. musculus 187cattgaccgt gtgggcggga 20 188 20 DNA M. musculus 188 aagcttcgggagatgggcag 20 189 20 DNA M. musculus 189 gggcagtcaa ggacaatccg 20 190 20DNA M. musculus 190 tatgcaacgt aactactgct 20 191 20 DNA M. musculus 191cagctcctct gagccatacc 20 192 20 DNA M. musculus 192 gccacaccagtcagagagcc 20 193 20 DNA M. musculus 193 tccgcgttct catgcttctc 20 194 20DNA M. musculus 194 gtacaacttc tgcggaagga 20 195 20 DNA M. musculus 195tgggttctgg ccacctcggg 20 196 20 DNA M. musculus 196 gtggcaagactactggactt 20 197 20 DNA M. musculus 197 agggtctctt cctctgggca 20 198 20DNA M. musculus 198 agtgctgtag agttcactgt 20

What is claimed is:
 1. A compound 8 to 80 nucleobases in length targetedto a nucleic acid molecule encoding LAR, wherein said compoundspecifically hybridizes with said nucleic acid molecule encoding LAR andinhibits the expression of LAR.
 2. The compound of claim 1 which is anantisense oligonucleotide.
 3. The compound of claim 2 wherein theantisense oligonucleotide comprises at least one modifiedinternucleoside linkage.
 4. The compound of claim 3 wherein the modifiedinternucleoside linkage is a phosphorothioate linkage.
 5. The compoundof claim 2 wherein the antisense oligonucleotide comprises at least onemodified sugar moiety.
 6. The compound of claim 5 wherein the modifiedsugar moiety is a 2′-O-methoxyethyl sugar moiety.
 7. The compound ofclaim 2 wherein the antisense oligonucleotide comprises at least onemodified nucleobase.
 8. The compound of claim 7 wherein the modifiednucleobase is a 5-methylcytosine.
 9. The compound of claim 2 wherein theantisense oligonucleotide is a chimeric oligonucleotide.
 10. A compound8 to 80 nucleobases in length which specifically hybridizes with atleast an 8-nucleobase portion of a preferred target region on a nucleicacid molecule encoding LAR.
 11. A composition comprising the compound ofclaim 1 and a pharmaceutically acceptable carrier or diluent.
 12. Thecomposition of claim 11 further comprising a colloidal dispersionsystem.
 13. The composition of claim 11 wherein the compound is anantisense oligonucleotide.
 14. A method of inhibiting the expression ofLAR in cells or tissues comprising contacting said cells or tissues withthe compound of claim 1 so that expression of LAR is inhibited.
 15. Amethod of treating an animal having a disease or condition associatedwith LAR comprising administering to said animal a therapeutically orprophylactically effective amount of the compound of claim 1 so thatexpression of LAR is inhibited.
 16. A method of screening for anantisense compound, the method comprising the steps of: a. contacting apreferred target region of a nucleic acid molecule encoding LAR with oneor more candidate antisense compounds, said candidate antisensecompounds comprising at least an 8-nucleobase portion which iscomplementary to said preferred target region, and b. selecting for oneor more candidate antisense compounds which inhibit the expression of anucleic acid molecule encoding LAR.
 17. The method of claim 15 whereinthe disease or condition is a hyperproliferative disorder.
 18. Themethod of claim 17 wherein the hyperproliferative disorder is cancer.19. The method of claim 15 wherein the disease or condition is ametabolic disorder.
 20. The method of claim 15 wherein the disease orcondition arises from aberrant apoptosis.